Dental  Library 


Gift  of 
Joseph  D.  Hodgen,  D.D.S. 


BACTERIOLOGY 


JOSEPft  p.  HODGEN  0.0.  S, 


BY    THE    SAME    AUTHOR, 

PATHOLOGY:    GENERAL    AND    SPECIAL 
For  Students  of  Medicine. 

Third  Edition.     32  Plates,  15  Text-figures.     10s.  6d.  net. 


SERUM  AND  VACCINE    THERAPY, 

BACTERIAL  THERAPEUTICS  AND 

PROPHYLAXIS,    BACTERIAL 

DIAGNOSTIC  AGENTS. 

Second  Edition. 
With  32  Figures.         Crown  8vo.         7s.  Gd.  net. 


Edited  by  PROF.  B.  T.  HEWLETT,  M.D.,  F.E.C.P. 
THE    CELL    AS    THE    UNIT    OF    LIFE 

and  other  Lectures  delivered  at  the  Royal  Institution, 
London,   1899-1902. 

AN  INTRODUCTION  TO  BIOLOGY 

By  the  late  ALLAN  MACFADYEN,  M.D.,  B.Sc. 

Fullerian  Professor  of  Physiology,  Royal  Institution,  London. 

With  16  Illustrations.         8vo.         75.  6d.  net. 


A  MANUAL 

OF 

BACTERIOLOGY 

CLINICAL  AND  APPLIED 

BY 
R.    TAN  N  E  R  |H  EWL  ETT 

M.D.,  F.R.C.P.,^¥lH.(Lond.) 

Professor  of  Bacteriology  in  the  University   of  London  ; 

Director  of  the  Bacteriological  Department,  King's 

College,     London ;     Director    of    Pathology, 

Seamen's  Hospital,  Greenwich  :  Lecturer 

on    Bacteriology,   London   School 

of   Tropical    Medicine 

FIFTH  EDITION 

JOSEPH  D.  I,  D.  D.  S. 

240  STOCKTON   STREET  Q  P^  ^ 


SAINT   LOUIS 
C.  V.  MOSBY  COMPANY 

1914 


Printed  in  C,>e<it  Britain 


PREFACE    TO    THE 
FIFTH  EDITION 

IN  this  edition  while  the  plan  of  the  book  remains  sub- 
stantially the  same,  the  text  has  been  completely  revised. 
Many  alterations  and  additions  have  been  made  in  the 
preliminary  chapters  dealing  with  the  structure  and 
classification  of  micro-organisms  and  with  the  methods 
of  cultivation,  isolation,  and  staining.  The  chapter  on 
the  side- chain  theory  and  immunity  reactions  has  been 
extended  and  new  details  respecting  anaphylaxis  inserted. 
Considerable  changes  have  been  effected  in  the  chapters 
dealing  with  particular  micro-organisms,  e.g.  the  Strepto- 
cocci, Anthrax,  Tubercle,  and  Leprosy  bacilli,  typhoid 
fever,  and  cholera.  More  space  is  devoted  to  the  Moulds, 
and  the  subjects  of  Sporotrichosis  and  Thrush  have  been 
transferred  to  this  section. 

In  the  chapter  dealing  with  the  Protozoa  the  cultivation 
of  the  Spirochaetes  has  been  inserted,  mention  is  made 
of  the  Luetin  reaction,  and  the  account  of  the  Wasser- 
mann  reaction  and  the  description  of  the  life  history  of 
the  rabbit  Coccidium  have  been  re-written.  The  pages 
dealing  with  Hydrophobia,  Vaccinia,  and  Malignant 
Disease  have  been  revised,  and  new  sections  on  Pellagra 
and  Rat-bite  Disease  have  been  inserted.  Further  details 


vi  PREFACE  TO  THE  FIFTH  EDITION 

are  given  on  the  purification  of  water,  and  the  bacteriology 
of  milk  and  foods  has  been  revised.  In  fact,  hardly  a 
page  remains  quite  the  same  as  previously,  and  it  is  hoped 
that  the  text  has  been  made  clearer  and  the  contained 
matter  brought  up  to  date. 

My  grateful  thanks  are  due  to  my  friends  and  colleagues, 
DR.  FRANK  TAYLOR,  upon  whom  the  labour  of  the  revision 
of  the  proof-sheets  has  largely  fallen,  and  MR.  J.  E. 
BARNARD,  who  has  furnished  some  new  photo- micrographs. 

R.  T.  H. 

UNIVERSITY  OF  LONDON]  KING'S]  COLLEGE 
May  1914 


PREFACE 

IN  the  following  manual  I  have  endeavoured  to  give  some 
account  of  those  portions  of  Bacteriology  which  are  of 
especial  interest  in  clinical  medicine  and  hygiene.  The 
preparation  of  tissues,  methods  of  culture,  descriptions  of 
pathogenic  organisms  and  their  detection,  the  examina- 
tion of  water,  etc.,  have  therefore  been  given  at  some 
length.  As  it  would  be  impossible  in  the  space  at  my 
disposal  to  include  everything  relative  to  the  subject,  a 
selection  has  had  to  be  made,  and  such  details  as  the 
celloidin  method,  Loffler's  stain  for  flagella,  the  strictly 
animal  parasitic  diseases  (with  a  few  exceptions),  etc., 
have,  among  others,  been  omitted. 

At  the  end  of  the  sections  dealing  with  the  pathogenic 
organisms  which  attack  man,  some  directions  have  been 
given  for  the  bacteriological  clinical  diagnosis  and  exami- 
nation, but  these  are  in  no  way  exhaustive ;  in  fact,  it 
would  not  be  possible  in  a  short  work  to  give  a  scheme 
of  examination  which  would  cover  every  case.  These 
directions  will  also  render  the  book  of  service  in  the 
laboratory,  while  I  venture  to  hope  that  the  details  given 
in  the  Appendix  on  the  use  of  the  remedies  and  diagnostic 
agents  of  bacterial  origin  may  be  of  value  to  the 
practitioner. 

I  have  to  thank  ME.  PEYTON  BEALE,  DR.  LAMBERT  LACK, 


viii  PREFACE 

and  ME.  F.  J.  TANNEK,  for  suggestions  and  criticisms,  and 
the  last-named  gentleman  for  the  aid  he  has  freely  given 
me  in  the  revision  of  the  proof-sheets.  I  am  also 
indebted,  indirectly,  in  many  ways  to  my  colleagues, 
DR.  MACFADYEN  and  MR.  FOULERTON.  My  thanks  are  due 
to  MR.  J.  BARNARD  and  to  MR.  FRANK  STRATTON  respec- 
tively for  the  photo-micrographs  and  original  drawings, 
while  for  the  eight  borrowed  illustrations  blocks  have 
been  kindly  lent  by  Messrs.  BAIRD  and  TATLOCK,  and 
Messrs.  SWIFT  and  SON. 

May  1898. 


CONTENTS 


CHAPTER  PAGE 

INTRODUCTION  1 

I.  THE  NATURE,  STRUCTURE,  AND  FUNCTIONS 
OF  THE  BACTERIA:  THEIR  CLASSIFICATION, 
GENERAL  BIOLOGY,  AND  CHEMISTRY— BAC- 
TERIA AND  DISEASE  8 

II.  METHODS     OF     CULTIVATING     AND     ISOLATING 

ORGANISMS  44 

III.  THE  PREPARATION  OF  TISSUES  AND  ORGANISMS 

FOR  STAINING  AND  MOUNTING— STAINING  AND 
STAINING  METHODS  86 

IV.  METHODS    OF   INVESTIGATING    MICROBIAL    DIS- 

EASES—THE INOCULATION  AND  DISSECTION 
OF  ANIMALS— HANGING-DROP  CULTIVATION— 
INTERLAMELLAR  FILMS— THE  MICROSCOPE  1 1  ; 

V.  INFECTION— VEGETABLE  AND  ANIMAL  PARA- 
SITES—THE INFECTIVE  PROCESS— ANTI- BODIES 
—ANTI-SERA  AND  ANTITOXINS— IMMUNITY— 
OPSONINS  144 

VI.  SUPPURATION  AND  SEPTIC  CONDITIONS  223 

VII.  ANTHRAX  251 

VIII.  DIPHTHERIA  265 

Diphtheria  in  England — The  Diphtheria  Bacillus — The 
Pseudo- Diphtheria  Bacillus — Clinical  Diagnosis — The 
Xerosis  Bacillus — Diphtheritic  Affections  of  Birds  and 
Animals 

IX.  "  ACID-FAST  "         BACILLI— TUBERCULOSIS— LEP- 
ROSY—THE  SMEGMA  BACILLUS— GLANDERS  299 

X.  TYPHOID  FEVER  —  PARA  -  TYPHOID  FEVER  — 
BACILLUS  ENTERITIDIS  AND  THE  GART- 
NER GROUP— SWINE  FEVER— BACILLUS  DYSEN- 
TERIC—BACILLUS COLI  351 

XI.  BUBONIC  PLAGUE— CHICKEN   CHOLERA— MOUSE 

SEPTICAEMIA  391 

ix  b 


x  CONTENTS 

CHAPTER  PAGE 

XII.  PNEUMONIA,      INFLUENZA,     AND     WHOOPING-     406 
COUGH 

XIII.  ANAEROBIC      ORGANISMS  — TETANUS  — MALIG- 

NANT (EDEMA  —  BACILLUS  BOTULINUS  — 
BACILLUS  WELCHII— BACILLUS  CADAVERIS 
SPOROGENES— BLACK  QUARTER— CLOSTRI- 
DIUM  BUTYRICUM  419 

XIV.  ASIATIC   CHOLERA— SPIRILLUM   METCHNIKOVI 

—SPIRILLUM  OF  FINKLER  AND  PRIOR— SPI- 
RILLUM TYROGENUM— SPIRILLUM  RUBRUM  433 

XV.  STREPTOTHRIX  INFECTIONS— ACTINOMYCOSIS 
— MYCETOMA— LEPTOTHRIX  BUCCALIS— CLA- 
DOTHRIX  DICHOTOMA  —  MYCOSIS  TONSIL- 
LARIS  450 

XVI.  THE  SACCHAROMYCETACE/E  461 

The  Yeasts — The  Pathogenic  Yeasts — Saccharomyces 
and  Torulae — Yeasts  and  Fermentation 

XVII.  THE    HYPHOMYCETES  —  ASPERGILLOSIS  —  SPO- 

ROTRICHOSIS— THRUSH— RINGWORM  469 

XVIII.  THE  PROTOZOA  480 

The  General  Structure  of  the  Protozoa — Pathogenic 
Amoebae — Trypanosomata — Leishman-Donovan  Body 
— Spirochaetae — Syphilis — Coccidia — Malaria 

XIX.  SCARLET  FEVER— HYDROPHOBIA— INFANTILE 
PARALYSIS— TYPHUS  FEVER— YELLOW  FEVER 
—DENGUE— PHLEBOTOMUS  FEVER— VACCINIA 
AND  VARIOLA— MALIGNANT  DISEASE  533 

XX.  SOME  DISEASES  NOT  PREVIOUSLY  REFERRED 
TO,  WITH  A  DISCUSSION  OF  THEIR  CAUSA- 
TION—MICRO-ORGANISMS OF  THE  SKIN  AND 
MUCOUS  MEMBRANES  556 

XXI.  THE  BACTERIOLOGY  OF  WATER,  AIR,  AND 
SOIL,  AND  THEIR  BACTERIOLOGICAL  EX- 
AMINATION —  SEWAGE  —  BACTERIOLOGY  OF 
MILK  AND  FOODS  572 

Some  of  the  Commoner  Organisms  found  in  the  Air, 
Water,  and  Soil 

XXII.  DISINFECTION  623 

Heat — Steam  Disinfection — Chemical  Disinfectants — 
Theory  of  Disinfection — Methods  of  determining  Dis- 
infectant Power 

FRENCH     WEIGHTS     AND     MEASURES     AND     THEIR 

ENGLISH  EQUIVALENTS— SOLUBILITIES  648 

INDEX  649 


LIST   OF   PLATES 

PLATE 

I.  PHAGOCYTOSIS  AND  M.  PYOGENE8           to  face  p.  228 

II.  STREPTOCOCCUS  PYOGENES  „  232 

III.  THE  MENINGOCOCCUS  AND  GONOCOCCUS  „  244 

IV.  ANTHRAX  „  252 

V.  ANTHRAX  „  254 

VI.  DIPHTHERIA  „  268 

VII.  THE  HOFMANN  BACILLUS  AND  VINCENT'S 

ANGINA  „  288 

VIII.  THE  TUBERCLE  BACILLUS  „  302 

IX.  THE  TUBERCLE  BACILLUS  „  310 

X.  LEPROSY  AND  B.  SMEGMAT1S  „  352 

XI.  B.  MALLEI  AND  GLANDERS  NODULE  „  344 

XII.  BACILLUS  TYPHOSUS  „  354 

XIII.  B.  TYPHOSUS  AND  B.  COLI  .,  382 

XIV.  PLAGUE  „  392 
XV.  B.  PESTIS  AND  CHICKEN  CHOLERA  „  394 

XVI.  DIPLOCOCCUS  PNEUMONIA  „  408 

XVII.  B.  TETANI  AND  B.  WELCH II  ,.  428 
XVIII.  SPIRILLUM  CHOLERA  AND  CULTURES 

OF  SPIRILLA  „  434 

XIX.  ACTINOM  YCOSIS  BO  VIS  AND  M  YCETOMA  452 


xii  LIST  OF  PLATES 

PLATE 

XX.  ACT  I  NO  MYCOSIS  HO  MINIS  to  face  p.  454 

XXL  TRYPANOSOMA  GAMBIENSE  AND 
8PIROCHAETA  RECURRENT1S  (OBER- 
MEIERI)  „  494 

XXII.  TREPONEMA  PALLIDUM  „  496 

XXflT.  TREPONEMA  PALLIDUM  AND  COCCI- 

DIUM  OV I  FORME  „  498 

XXIV.  THE  MALARIA  PARASITE  „  520 

XXV.  TERTIAN  "ROSETTE"  AND  HALTERI- 

DIUM  DANILEWSKYI  „  522 

XXVI.  PIROPLA8MA  CAN  IS  AND  HMMOCYSTI- 

DIUM  OF  COBRA  530 


ERRATUM 

On  p.  230,  Table,  Col.  1,  for  Micrococcus  epidermis  read  Micrococcus 
epidermidis 


A  MANUAL  OF 

BACTERIOLOGY 


INTRODUCTION 

BACTERIOLOGY  is  a  branch  of  Biology  which  deals  with 
the  study  of  Micro-organisms,  particularly  the  minute 
vegetable  ones  known  as  Bacteria.  The  scope  of  bacterio- 
logy is  difficult  to  define  exactly,  for  the  term  is  often 
used  in  a  comprehensive  sense  equivalent  to  micro- 
pathology,  or  even  micro-biology,  and  all  investigations 
connected  with  micro-organisms,  animal  and  vegetable, 
may  be  included  under  it.  So  extensive,  however,  has 
the  subject  become  that  the  animal  micro-organisms  are 
now  being  studied  as  a  separate  branch,  PROTOZOOLOGY. 
Bacteriology  deals  with  micro-organisms  particularly  in 
their  relation  to  processes — disease,  fermentation,  putre- 
faction, and  the  like — while  the  study  of  their  structure, 
functions,  and  life-history  belongs  to  the  botanist  and 
zoologist.  There  is  no  space  in  this  work  to  enter  into  the 
history  of  the  science,  but  the  names  of  Leeuwenhoek 
(1675),  Miiller  (1786),  Schwann  (1837),  Cohn,  Pasteur, 
Lister,  and  Koch  will  ever  hold  an  honourable  place  in  its 
annals. 

The  study  of  micro-organisms  must  always  be  of 
importance  in  general  biology,  for  their  vital  phenomena 
are  comparatively  simple,  and  shed  light  on  the  more 
complex  processes  occurring  in  the  higher  orders  of  living 

1  I 


2  INTRODUCTION 

beings.  Weismann  based  his  theory  of  heredity  on  the 
fundamental  conception  of  the  immortality  of  these 
unicellular  organisms.  Excluding  accidents,  they  are 
immortal — they  reproduce  themselves  by  a  process  of 
simple  division,  an  individual  dividing,  and  two  daughter 
forms  taking  the  place  of  the  original  parent  one,  and 
although  the  parent  has  disappeared  yet  there  has  been  no 
death,  no  dissolution  ;  its  protoplasm  or  living  material 
is  still  existent  in  its  progeny  and  is  immortal,  since  this 
process  of  reproduction  apparently  may  go  on  indefinitely. 
Moreover,  the  study  of  the  mutability  and  possible  trans- 
formation of  species  of  micro-organisms  is  likely  to  throw 
light  on  the  theory  of  evolution.  Organisms  such  as 
bacteria  multiply  so  rapidly  that  fifty  or  sixty  generations 
may  develop  in  thirty  hours,  a  number  which  would  take 
years  to  attain  if  even  the  most  rapid  breeder  among 
mammals  were  the  subject  of  experiment,  and  as  they 
occur  in  vast  numbers  the  opportunity  for  variation  is 
extensive.  These  are  some  of  the  relations  which  micro- 
organisms have  with  general  biology. 

In  what  may  be  termed  the  economy  of  nature  micro- 
organisms are  all-important ;  without  them  there  would 
be  no  putrefaction,  no  decay,  and  the  dead  remains  of 
animal  and  vegetable  life  would  accumulate  and  encumber 
the  earth,  which  would  become  barren  for  the  want  of 
the  organic  matter  originally  derived  from  it,  but  of  which 
there  was  no  return.  In  fact  the  higher  plants,  and 
indirectly,  therefore,  animals  also,  are  dependent  for  their 
existence  upon  the  presence  of  bacteria  in  the  soil,  which 
break  up  and  render  assimilable  complex  substances  used 
as  manures. 

The  question  of  life,  animal  and  vegetable,  without 
bacterial  activity  is  an  important  and  interesting  one. 
It  would  seem  from  the  experiments  of  Duclaux1  that  the 
1  Comp.  Rend.,  t.  100,  p.  66, 


INTRODUCTION  3 

higher  plants  in  ordinary  circumstances  are  unable  to 
obtain  nutriment  unless  the  complex  compounds,  pro- 
teins, urea,  and  other  nitrogenous  bodies,  which  form 
the  important  constituents  of  many  manures,  are  broken 
down  into  simpler  ones  through  the  agency  of  bacteria. 
He  sowed  seeds  in  sterile  soil  free  from  nitrates,  nitrites, 
and  ammonia,  which  was  plentifully  watered  with  sterile 
milk  and  solutions  of  sugar  and  starch.  No  changes 
occurred  in  these  substances,  the  seeds  lost  weight,  and 
the  seedlings  dwindled  and  died.  As  regards  the  higher 
animals  various  views  have  been  expressed.  Pasteur 
considered  that  their  life  also  would  probably  be  im- 
possible without  the  presence  of  bacteria  in  the  intestinal 
tract.  Nencki  expressed  the  opinion  that  this  idea  of 
Pasteur's  was  an  erroneous  one,  and  his  experiments  in 
conjunction  with  Macfadyen  and  Sieber1  showed  that  any 
considerable  decomposition  of  the  food  by  bacteria  first 
takes  place  in  the  large  intestine,  and  that  the  digestive 
juices  alone,  without  the  co-operation  of  bacteria,  are  able 
to  prepare  the  constituents  of  the  food  for  absorption. 
Nuttall  and  Thierfelder  obtained  unborn  guinea-pigs  by 
Caesarian  section  with  antiseptic  precautions,  and  after- 
wards kept  them  in  a  sterile  environment  and  fed  them 
on  sterilised  food.  Not  only  did  the  animals  live,  but 
they  were  even  in  a  more  thriving  condition  than  those 
naturally  brought  up.  The  intestinal  tract  was  found  to 
be  sterile  on  the  eighth  day.  Schottelius,  however,  found 
that  chickens  reared  on  sterile  food  were  retarded  in 
development,  and  experiments  by  Moro  on  turtle  larvae 
point  to  the  same  conclusion,  viz.  that  intestinal  bacteria 
are  necessary  for  normal  nutrition.  On  the  other  hand, 
Cohendy2  finds  that  chickens  can  be  reared  perfectly  well 
without  the  presence  of  bacteria.  Levin  found  that  the 

1  Journ..  of  Anat.  and  PhysioL,  xxv,  p.  390.  . 

2  Ann.  de  rinst.  Pasteur,  xxvi,  1912,  p.  106. 


4  INTRODUCTION 

intestinal  tract  in  many  Arctic  animals — the  polar  bear, 
reindeer,  seal,  eider  duck,  etc. — is  generally  sterile,  and  in 
these  instances,  therefore,  bacteria  are  not  required  for 
normal  nutrition. 

Commercially,  micro-organisms  are  of  the  utmost 
importance.  Without  them  there  would  be  no  fermen- 
tation, and  the  wine,  beer,  and  indigo  industries,  the 
ripening  of  cheese  and  tobacco,  and  many  like  processes 
would  be  non-existent.  From  a  financial  aspect  also 
micro-organisms  cannot  be  ignored,  for  many  of  the  so- 
called  "  diseases  "  of  beer  and  wine,  which  often  occasion 
great  loss,  are  due  to  the  entrance  of  adventitious  forms, 
while  the  silk  industry  and  sheep  farming  in  France  were 
once  threatened  with  extinction  owing  to  the  ravages  of 
pebrine  and  of  anthrax  respectively,  but  through  the 
genius  of  Pasteur  were  restored  to  their  former  prosperity. 
There  is  no  need  to  emphasise  the  importance  of  micro- 
organisms frorn  a  medical  and  hygienic  point  of  view, 
but  the  fact  may  be  recalled  that  sixty  years  ago  the 
mortality  after  operations  was  very  high,  and  that  40  per 
cent,  of  these  deaths  were  caused  by  pyaemia,  septicaemia, 
and  hospital  gangrene,  conditions  which  are  due  to  the 
entrance  of  micro-organisms,  and  which  are  now  almost 
preventable,  thanks  to  the  antiseptic  system  introduced 
by  Lord  Lister. 

The  theory  of  spontaneous  generation  or  abiogenesis  is 
intimately  connected  with  the  study  of  bacteria.  The 
putrefaction  of  animal  and  vegetable  fluids  even  after 
boiling,  and  the  growth  in  them  of  minute  living  forms, 
were  held  by  many  to  be  a  sure  proof  of  the  development 
of  life  from  inanimate  matter,  of  the  spontaneous  genera- 
tion of  the  living  from  the  non-living.  A  succession  of 
investigators,  however,  showed  (1)  that  if  the  fluids  be 
boiled  sufficiently  long,  and  be  then  sealed  up  so  as  to 
prevent  the  access  of  air,  they  do  not  undergo  putre- 


INTRODUCTION  5 

faction  ;  (2)  that  the  sealing  up  may  be  dispensed  with, 
provided  the  air  be  first  filtered  through  cotton-wool  before 
being  admitted  to  the  flasks  ;  (3)  that  even  the  cotton- 
wool is  not  needed  if  the  air  be  passed  slowly  through  a 
long  and  tortuous  channel,  so  as  to  deposit  its  solid 
particles.  Tyndall  showed  that  putrescible  fluids  may  be 
exposed  in  open  vessels  in  a  closed  chamber  in  which  the 
air  has  been  undisturbed  for  some  time  and  its  solid 
particles  thereby  deposited  on  the  walls  of  the  chamber, 
which  had  been  smeared  with  glycerin  ;  he  also  proved 
that  vegetable  infusions  and  the  like,  which  putrefy  after 
having  been  boiled  for  ten  minutes,  do  not  do  so  if  the 
boiling  be  repeated  on  two  or  three  successive  days,  and 
explained  this  by  the  supposition  that  while  the  fully 
developed  bacteria  are  destroyed  by  the  first  boiling, 
their  more  resistant  spores  remain  alive,  but  these  on 
being  left  for  twenty-four  hours  germinate  into  the  less 
resistant  bacterial  forms,  which  are  destroyed  by  the 
second  boiling,  and  by  the  repetition  of  the  process  com- 
plete sterilisation  may  ultimately  be  obtained.  It  is 
this  process  of  "  discontinuous  sterilisation,"  as  it  is 
termed,  which  is  employed  by  the  bacteriologist  for  the 
preparation  of  sterile  culture  media.1 

The  occurrence  of  abiogenesis  (or  as  he  prefers  to  term 
it,  "  archebiosis  ")  is  still  maintained  by  Bastian.  He 
claims  that  certain  saline  solutions  which  have  been  boiled 
or  even  heated  above  the  boiling-point  in  sealed  tubes 
after  a  time  show  the  development  of  various  living 
organisms,  including  bacteria  and  yeasts.2 

Dunbar,3  as  the  result  of  a  series  of  experiments  con- 

1  The  writer  believes  that  this  explanation  is  only  partially  true, 
and  would  ascribe  some  of  the  sterilising  effect  of  repeated  heatings 
simply  to  the  injurious  action  of  alternate  heating  and  cooling. 

2  See  various  papers  in  the  Proc.  Roy.  Soc.  Lond.  ;   The  Evolution  of 
Life,  Methuen,  1907  ;   and  Proc.  Roy.  Soc.  Med.  1913. 

3  See  Journ.  Roy.  Inst.  Pub.  Health,  vol.  xv,  No.  11,  1907,  p.  679. 


6  INTRODUCTION 

ducted  over  a  long  period  and  with  every  care  to  prevent 
contamination,  has  come  to  the  conclusion  that  the 
bacteria  are  not  an  independent  group  of  organisms,  but 
that  the  bacteria,  yeasts,  and  moulds  are  stages  in  the 
life-history  of  green  algae.  The  observations  were  carried 
out  both  by  culture  methods  and  by  microscopical  examina- 
tion. A  culture  of  a  single-celled  alga  belonging  to  the 
Palmellacea  was  obtained,  but  by  modifying  the  culture 
medium  in  which  a  pure  culture  of  the  alga  was  growing, 
by  the  addition  of  acid,  of  alkali,  or  of  traces  of  copper 
salts,  other  organisms,  generally  bacteria,  occasionally 
moulds  and  yeasts,  and  even  spirochaetes,  made  their 
appearance.  Granting  that  there  is  no  flaw  in  the  experi- 
mental methods,  and  every  care  seems  to  have  been  taken 
to  exclude  contamination,  etc.,  the  results  are  susceptible 
of  another  explanation,  viz.  that  the  secondary  growths 
were  derived  by  transformation  of  the  algal  cells,  in  fact 
by  the  phenomenon  of  heterogenesis  which  has  been 
claimed  by  Bastian  to  occur. 

Undoubtedly  bacteria  exhibit  variations  and  mutations, 
not  only  in  morphology  (see  p.  16)  but  also  in  function. 
Thus  pathogenic  organisms  may  become  non-pathogenic, 
and  Twort  has  succeeded  in  training  B.  typhosus  to  fer- 
ment lactose,  which  ordinarily  it  does  not.  Some  experi- 
ments by  Horrocks  suggest  that  the  B.  typhosus  may,  by 
symbiosis  with  B.  coli,  be  converted  into  B.  alcaligenes, 
and  Revis  has  found  many  variations  occur  with  coliform 
organisms  as  a  result  of  cultivating  in  malachite  green 
media,  etc.1 

As  a  result  of  exposure  of  sporing  anthrax  to  ultra-violet 
rays,  Mme.  Henri  2  states  that  stable  coccoid  and  Gram- 
negative,  thin  filamentous  forms  are  obtained. 

1  Proc.  Roy.  Soc.  Lond.,  B,  vol.  85,  p.  192,  and  vol.  86,  p.  373  ;  Cenlr. 
/.  Bakt.,  Abt.  II,  ]912  and  "1913. 

2  Comp.  Rend.  Acad.  Sc.,  vol.  158,  No.  14,  1914,  p.  1032. 


INTRODUCTION  7 

Minchin  in  a  presidential  address  to  the  Quekett  Micro- 
scopical Club  points  out  that  syngamy  (sexual  reproduction, 
e.g.  conjugation)  is  of  the  greatest  importance  in  preserving 
differentiation  of  species,  and  that  without  it  a  species 
will  tend  to  break  up  into  races.  It  therefore  follows 
that  there  are  no  true  species  among  organisms  of  the 
bacterial  grade,  if  it  be  true,  as  is  usually  held,  that 
syngamy  does  not  occur  amongst  them,  and  the  so-called 
species  of  bacteria  are  to  be  regarded  as  mere  races  or 
strains  capable  of  modification  in  any  direction. 

While  much  progress  has  been  made  during  the  last 
two  or  three  decades,  a  vast  amount  still  remains  to 
be  done.  We  have  only  touched  the  fringe  of  the  explana- 
tion of  the  perplexing  problems  of  susceptibility  and 
immunity,  and  of  the  important  question  of  cure  in,  and 
prevention  of,  infective  diseases,  while  the  chemistry  of 
the  products  of  bacterial  activity  is  still  in  its  infancy. 

The  literature  of  Bacteriology  has  now  become  somewhat  exten- 
sive. In  the  following  pages  references  to  original  papers  have 
been  freely  introduced,  many  of  which  contain  a  more  or  less  full 
bibliography  on  the  subject  referred  to,  so  that  further  information 
may  be  obtained  if  required.  Kolle  and  Wassermann's  Handbuch 
der  Pathogenen  Mikroorganismen,  ed.  ii,  is  the  most  encyclopedic 
work  on  pathological  bacteriology  yet  published. 


CHAPTER  1 

THE  NATURE,  STRUCTURE,  AND  FUNCTIONS  OF  THE 
BACTERIA:  THEIR  CLASSIFICATION,  GENERAL  BIO- 
LOGY, AND  CHEMISTRY— BACTERIA  AND  DISEASE 

THE  Bacteria  or  Schizomycetes  ("  fission  fungi ")  are 
minute  vegetable  organisms  for  the  most  part  unicellular 
and  devoid  of  chlorophyll,  which  multiply  by  simple  trans- 
verse division  or  fission  ;  this  distinguishes  them  from  the 
yeasts,  in  which  multiplication  takes  place  by  budding  or 
gemmation.  A  certain  number  of  filamentous  forms  are 
also  included,  serving  to  connect  the  unicellular  ones  with 
the  multicellular  true  fungi.  The  "  fission  plants  "  may 
be  placed  in  a  sub-kingdom,  the  Schizophyta,  which 
may  be  divided  into  two  classes  :  Class  I,  Schizophycese, 
the  blue-green  algae,  and  Class  II,  Schizomycetes,  the 
bacteria. 

The  unicellular  plants  are  sometimes  termed  the  "  Proto- 
phyta."  It  must  be  understood  that  there  are  connecting 
links  between  the  different  groups,  and  that  there  is  no 
sharp  line  of  demarcation  between  them. 

The  relation  of  the  bacteria  to  other  lower  plants  is 
shown  in  the  following  scheme  (p.  9)  : 

The  Bacteria  have  been  supposed  to  have  affinities  with 
the  Fungi,  with  the  Protozoa,  or  with  the  Cyanophycese. 
There  is  little  or  no  evidence  to  connect  them  with  the  first 
two  groups  and  not  much  with  the  last  one,  though  the 
resemblances  here  are  greater.  Though  usually  regarded 
as  simple  forms,  the  Bacteria  display  considerable  morpho- 

8 


STRUCTURE  OF  BACTERIA  9 

logical    and   structural  differentiation   and   physiological 
complexity  and  are  by  no  means  primitive  forms. 

The  size  of  the  bacteria  is  variable,  but  they  are  all 
microscopic,  measuring  0-3/x  to  30-40^  in  diameter  or 
in  length.1  Their  shape  likewise  is  very  different  in  the 
different  species  ;  some  are  spherical,  others  ovoid,  others 

Relation  of  Bacteria  to  Lower  Plants 

Thallophyta  (lower  plants  without  fibro-vascular  bundles,  and  with 
no  distinction  between  root  and  stem) 


Forms  with  chlorophyll  Forms  without  chlorophyll 

(Algae,  desmids,  etc.)  | 

Multicellular.     Spores  in  Unicellular.     Spores  frequently 

differentiated  cells  or  spore-  absent.     Spore-bearing  cells  not, 

bearing  organs.     Generally  or  but  slightly,  differentiated. 

a  sexual  method  of  reproduction  Sexual  reproduction  usually  absent 

I  I 

The  true  Fungi  (Eumycetes) 

including  moulds  (Hypho-         Reproduction  Reproduction 

mycetes)  by  fission  by  budding 

I  I 

The  Schizomycetes       The  Saccharomycetes 
or  Bacteria  or  Yeasts 

rod-shaped  or  filamentous,  while  in  some  the  rod  or  fila- 
ment is  twisted  into  a  spiral.  The  end  of  the  cell  is 
occasionally  almost  rectangular,  but  is  usually  more  or 
less  rounded  ;  it  is  probably  never  pointed  except  in 
the  Spirochaeta?,  if  these  be  true  Bacteria  (see  p.  18). 
The  bacterial  cell  consists  of  a  cell  -membrane  enclosing 
the  transparent,  more  or  less  structureless  living  matter 
or  protoplasm,  the  cell-plasma  or  cytoplasm.  Biitschli 
has  described  the  bacterial  plasma  as  having  a  reticular 
structure,  but  in  the  young  cell  this  is  probably  either 
an  artifact  or  a  "  false  image  "  due  to  faulty  illumination  ; 
the  most  that  can  be  seen  is  a  fine  granulation.  The 

1  p.  =  micron  =  0-001  mm. 


10  A  MANUAL  OF  BACTERIOLOGY 

protoplasm  frequently  contains  granules  composed  of 
fatty  or  protein  matter,  pigment,  and  in  some  species  of 
sulphur ;  occasionally  certain  granules  stain  blue  with 
iodine.  In  some  species  "  metachromatic  "  granules  occur, 
chiefly  at  the  poles  ;  these  stain  red  or  pink  with  many 
blue  dyes,  e.g.  methylene  blue,  are  composed  of  nucleic 
acid  combined  with  an  organic  base  and  are  to  be  regarded 
as  non-living  reserve  material  (Dobell). 

In  the  past  many  have  regarded  the  bacteria  as  enucleate 
cells.  This  is  probably  incorrect,  and  Dobell  finds  that 
all  bacteria  investigated  possess  a  nucleus  which  may  be 
'in  the  form  of  discrete  granules  (chromidia),  a  filament  of 
variable  configuration,  one  or  more  relatively  large  aggre- 
gated masses  of  nuclear  substance,  or  a  system  of  irregularly 
branched  or  bent  short  strands,  rods,  or  networks,  and 
probably  also  in  the  vesicular  form.  The  granules  observed 
by  Rowland  to  take  part  in  cell  division  (see  below)  and 
staining  with  roseine  are  probably  chromidia. 

The  cell-membrane  is  usually  invisible,  but  if  the  cell 
is  treated  with  salt -solution  (2-5  per  cent.)  plasmolysis 
takes  place,  the  protoplasm  shrinking  away  from  the 
membrane,  which  then  becomes  visible.  It  can  also  be 
stained  in  vivo  with  very  dilute  solutions  of  roseine.  The 
cell-membrane  sometimes  becomes  thickened,  swollen, 
and  gelatinous  on  its  outer  surface,  forming  a  layer  or 
so-called  "  capsule  "  around  the  organism.  The  clear 
spaces  frequently  seen  around  bacteria  in  dried  and  stained 
preparations,  especially  in  those  from  blood  and  lymph, 
are  generally  artifacts  and  not  true  capsules.  In  Clado- 
ihrix  and  some  other  forms  the  cell-membrane  becomes 
hardened,  leading  to  the  production  of  a  firm  sheath. 
When  bacteria  assume  the  resting  stage  groups  of  them 
adhere  together  in  a  jelly-like  matrix,  forming  what  is 
known  as  a  "  zooglcea." 

The  chemical  composition  of  bacteria  varies  much,  not 


BROWNIAN  MOVEMENT  11 

only  in  different  species,  but  even  in  the  same  species 
when  grown  on  different  nutrient  media.  All  bacteria 
contain  proteins,  lipoid  substances,  and  salts.  Bacterial 
protein,  according  to  Nencki,  differs  from  ordinary  protein 
matter  in  not  being  precipitated  by  alcohol  and  in  not 
containing  sulphur ;  it  was  termed  by  him  "  myko- 
protein."  This  does  not  appear  to  be  the  case  with  the 
proteins  obtained  by  grinding  bacterial  cells,  which  seem 
to  agree  with  other  proteins  in  heat-coagulation,  etc. 

The  proteins  are  mainly  globulins  and  nucleo-proteins. 
The  cell  wall  is  relatively  insoluble,  and  generally  consists 
chiefly  of  a  material  like  ckitin,  and  not  of  cellulose  ;  in 
this  respect  bacteria  resemble  animal  rather  than  vegetable 
cells.  Carbohydrates  are  generally  scanty.  Spores  differ 
from  the  parent  cells  in  containing  a  larger  proportion  of 
solids  and  less  water. 

All  species  of  bacteria,  but  especially  the  smaller  ones, 
when  suspended  in  a  fluid  exhibit  what  is  known  as. 
Brownian  movement,  consisting  of  an  oscillation  with 
some  amount  of  rotation  about  a  fixed  point,  but  there 
is  little  actual  movement  of  translation,  unless  due  to 
flotation.  This  Brownian  movement  is  physical  and  not 
vital  in  origin,  and  occurs  with  all  fine  particles  suspended 
in  a  fluid,  and  must  be  clearly  distinguished  from  a  true 
vital  motility.1  Some  bacteria  are  always  motionless, 
others  are  more  or  less  motile,  but  these,  too,  have  a 
resting  stage.  For  motility  to  occur  the  cells  must  be 
young,  and  the  conditions  favourable  to  growth  and 
development.  Motility  is  due  to  delicate  protoplasmic 
threads  termed  "  flagella  "  connected  with  the  outer  layer 
of  the  cell  protoplasm  ;  these  vibrate  to  and  fro  and 
propel  the  organism  through  the  medium.  A  cell  will, 

1  Brownian  movement  is  due  to  "  the  incessant  movements  of  the 
molecules  of  the -liquid  which,  striking  incessantly  the  observed  par- 
ticles, drive  them  about  irregularly  through  the  fluid  "  (Perrin). 


12  A  MANUAL  OF  BACTERIOLOGY 

however,  move  indifferently  in  either  direction ;  if  a 
motile  organism  be  watched  it  will  often  be  seen  to  proceed 
rapidly  in  one  direction,  stop,  and  then  return  without 
turning  round.  The  flagella  are  not  visible  in  the  living 
state,  unless  dark  ground  illumination  be  used,  nor  by  the 
ordinary  methods  of  staining,  unless  previously  treated 
with  a  mordant,  and  are  extremely  liable  to  be  broken 
off.  They  vary  considerably  in  number  and  in  length ; 
some  organisms  have  but  a  single  flagellum  at  one  pole 
(monotrichic),  e.g.  Bacillus  pyocyaneus,  others  have  two 
or  more  flagella  forming  a  brush  or  tuft  (lophotrichic), 
e.g.  Spirillum  rubrum,  while  others  may  be  almost  entirely 
covered  with  them  (peritrichic),  e.g.  B.  typhosus ;  in  some 
the  flagella  are  short  and  straight,  and  in  others  long  and 
twisted.  The  motility  of  organisms  does  not  necessarily 
depend  directly  upon  the  number  of  flagella  they  possess, 
an  organism  with  a  few  flagella  often  being  more  active 
than  another  possessing  many,  and  some  are  apparently 
non-motile,  though  well-marked  flagella  can  be  demon- 
strated. Generally  speaking,  however,  an  organism  with 
several  flagella  will  be  more  motile  than  a  similar  form 
with  a  few. 

Darwin  says  :  "  In  looking  at  Nature  it  is  most  necessary 
never  to  forget  that  every  single  organic  being  may  be 
said  to  be  striving  to  the  utmost  to  increase  in  numbers," 
and  in  no  group  perhaps  of  the  animal  and  vegetable 
kingdoms  is  this  more  marked  than  among  the 
Bacteria.  Reproduction  is  probably  always  non-sexual, 
and  takes  place  in  two  ways — by  simple  division  or  fission 
and  by  spore  formation.  Schaudinn  described  an  apparent 
conjugation  in  one  species  (B.  flexilis)  and  Nadson  states 
that  in  a  few  species  sister  cells  conjugate  and  from  this 
conjugation  a  spore  arises.  Dobell,  however,  considers 
that  all  the  evidence  is  definitely  against  the  view  that  a 
sexual  process  occurs  at  any  stage  in  the  life -history  of 


REPRODUCTION  OF  BACTERIA  13 

Bacteria.  Reproduction  by  transverse  fission  is  common 
to  all  bacteria  ;  the  bacterial  cell  becomes  constricted  at 
its  middle  and  finally  separates  into  two  parts,  and  thus 
two  young  cells  take  the  place  of  the  parent  one  ;  repro- 
duction by  fission  is  therefore  also  an  increase  in  numbers. 
The  fission  is  always  transverse,  never  longitudinal,1  the 
rule  being  in  cell-division  that  the  new  membrane  is 
formed  in  the  most  economical  manner.  Longitudinal 
division,  on  the  other  hand,  is  comparatively  common 
among  the  Protozoa.  Previous  to  division  the  rod-forms 
become  elongated  and  the  spherical  ones  ellipsoidal,  and 
there  is  an  increase  in  the  number  of  the  roseine-staining 
granules,  partly  by  division  of  pre-existing  ones  and  partly 
by  new  formation.  The  constriction  in  the  majority  of 
cases  involves  and  passes  through  one  of  the  granules. 
In  the  monotrichous  and  lophotrichous  bacteria  it  is 
always  the  non-flagellated  end  of  the  dividing  cell  which 
bears  the  flagella  of  the  new  cell.  Under  favourable 
conditions  reproduction  may  be  very  rapid,  fission  occurring 
every  twenty  or  thirty  minutes  (Klein),  so  that,  the  increase 
being  in  a  geometrical  ratio,  the  number  of  individuals 
which  might  arise  from  a  single  bacterium  in  three  or  four 
days  is  almost  inconceivable,  and  would  en  masse  weigh 
thousands  of  tons  ;  fortunately  there  are  many  checks  to 
such  a  rapid  multiplication.  Frequently,  although  the 
protoplasm  divides,  the  division  of  the  cell-membrane  is 
incomplete,  resulting  in  a  loose  union  of  the  cells  with  the 
formation  of  a  pseudo-filament.  These  filaments  often 
become  much  curved  and  twisted,  forming  tangled  masses, 
owing  to  fission  taking  place  in  the  cells  in  the  middle  of 
the  filament  as  well  as  at  the  ends,  so  that  the  filaments 
have  to  become  curved  to  make  room  for  the  new  cells. 

1  Longitudinal  division  has  been  described  in  a  few  species,  but  its 
occurrence  is  so  rare  that  a  doubt  must  arise  as  to  whether  these  forms 
are  true  bacteria. 


14  A  MANUAL  OF  BACTERIOLOGY 

Reproduction  by  spore  formation  is  met  with  in  some 
species,  and  is  generally  described  as  being  of  two  kinds. 
In  the  first,  "  endogenous  "  spore  formation,  a  bright 
refractile  round  or  ovoid  body  is  formed  within  the  bacterial 
cell,  the  development  of  which  can  be  watched  under  the 
microscope.  Rowland  describes  the  process  of  spore 
formation  as  follows  :  Refractile,  oily-looking  droplets, 
which  do  not  stain  with  roseine,  appear  and  ultimately 
coalesce,  forming  the  spore.  The  cell-plasma  at  the  same 
time  diminishes  and  retracts  from  the  cell-membrane. 
The  roseine-staining  granules  increase  in  number  and 
aggregate  into  two  spherical  masses,  which  dispose  them- 
selves one  at  each  end  of  the  cell.  The  cell-membrane 
collapses  somewhat,  and,  when  the  spore  is  fully  formed, 
ruptures  transversely,  leaving  two  cup-shaped  receptacles, 
in  which  the  granules  and  remains  of  the  plasma  are  still 
recognisable.  Only  one  spore  develops  in  each  cell,  and 
the  spores  serve  to  perpetuate  the  race  when  it  is  threatened 
with  extinction  from  adverse  circumstances.  Each  spore 
consists  of  a  little  mass  of  protoplasm  enclosed  within  a 
very  tough  and  resisting  membrane,  which  tends  to  pre- 
serve its  vitality  even  under  unfavourable  conditions  ; 
for  spores  resist  the  action  of  desiccation  and  germicidal 
agents  to  a  much  greater  degree  than  the  fully  developed 
organisms.  Spores  vary  much  in  size  and  in  the  position 
they  occupy  within  the  bacterial  cell  in  the  different 
species  ;  their  diameter  is  usually  about  the  same  as  that 
of  the  cell  in  which  they  are  developed,  but  may  be  much 
greater,  and  in  position  they  may  be  central  or  terminal, 
and  sometimes  the  spore -bearing  cells  are  swollen  or 
club-shaped  ;  these  are  termed  "  clostridia."  Endospores 
are  still  unknown  in  a  large  number  of  species.  The 
second  variety  of  sporulation,  "  arthrospore  "  formation, 
is  of  doubtful  occurrence,  but  is  stated  to  take  place  as 
follows  :  Some  of  the  elements  formed  by  fission  are 


CLASSIFICATION  OF  BACTERIA  15 

slightly  larger,  more  refractile,  and  more  resisting  than 
their  fellows,  and  are  stated  to  have  the  properties  of 
spores.  Placed  in  favourable  circumstances,  the  spore  in 
either  case  germinates,  it  becomes  swollen  and  granular, 
and  loses  its  refractile  appearance  ;  a  slight  protuberance 
forms,  this  increases  in  size,  and  an  organism  similar 
to  the  parent  one  is  finally  reproduced ;  the  empty 
spore  membrane  at  first  frequently  encloses  one  ex- 
tremity, and  is  afterwards  cast  off.  In  certain  instances 
the  spore  germinates  without  casting  its  membrane, 
the  spore  membrane  becoming  the  cell-wall  of  the 
young  organism.  The  ellipsoidal  spores  of  the  B. 
anthracis  sprout  from  the  end,  those  of  B.  subtilis  from 
the  side  ("  polar "  and  "  equatorial  "  germination 
respectively). 

On  the  Morphology,  etc.,  of  the  Bacteria  see  Dobell,  Quart, 
Journ.  Micr.  Sci.,  vol.  56,  1911,  p.  395  (Bibliog.)  ;  Penau,  Comp. 
Rend.,  clii,  1911,  p.  53  ;  Prazmowski,  Bull.  Interned,  de  VAcad.  des 
Sci.  de  Cracovie,  No.  4,  B,  April  1913,  p.  105  (Bibliog.). 


Classification  of  the  Bacteria 

Many  classifications  of  the  Bacteria  have  been  proposed, 
but  none  can  be  said  to  be  strictly  scientific,  or  even 
satisfactory  from  the  standpoint  of  convenience.  A  some- 
what heterogenous  group  of  organisms  has  undoubtedly 
been  described  under  the  term  Bacteria,  and  many  forms 
exist  intermediate  between  those  unicellular  organisms 
with  and  those  without  chlorophyll,  so  that  a  hard  and 
fast  line  cannot  be  drawn.  Moreover,  bacterial  cells  are 
so  minute  that  only  a  few  broad  differences  can  be  observed 
in  the  morphology  and  reproductive  processes  of  different 
species,  and  therefore  ordinary  criteria  are  not  available 
for  the  classification  of  the  Bacteria. 

One  of  the  most  prominent  of  the  older  classifications 


16  A  MANUAL  OF  BACTERIOLOGY 

was  that  of  Cohn.     He  divided  the  Bacteria  into  four 
principal  groups  : 

I.  The  Sphserobacteria  or  spherical  forms. 
II.  The  Microbacteria  or  short  rod-forms. 

III.  The  Desmobacteria  or  long  rod-forms. 

IV.  The  Spirobacteria  or  spiral  forms. 

Zopf's  classification  (1885)  has  many  points  to  commend 
it,  but  is  largely  based  on  the  occurrence  of  pleomorphism. 
By  pleomorphism  is  meant  a  variation  in  the  form  of  an 
organism  during  its  life-cycle,  a  coccus,  for  example, 
growing  into  a  rod,  or  a  straight  rod  becoming  a  spiral. 
In  a  peach-coloured  bacterium  examined  by  Lankester, 
coccoid,  rod,  filamentous,  and  spiral  forms  occurred,  and 
the  doctrine  of  pleomorphism  received  considerable  support 
from  his  work,  though  it  may  be  questioned  whether  he 
was  working  with  pure  cultures.  Be  that  as  it  may,  a 
certain  amount  of  pleomorphism  undoubtedly  occurs  in 
some  organisms.  In  the  colon,  typhoid,  and  plague 
bacilli,  for  example,  the  rods  may  sometimes  be  so  short 
as  to  be  almost  cocci,  while  at  others  they  are  well-marked 
rods  and  even  filaments  (see  also  p.  6).1  The  following  is 
an  outline  of  Zopf's  classification,  the  Bacteria  being 
divided  into  four  principal  groups  or  families,  which  again 
are  subdivided  into  smaller  groups  or  genera  : 

Family   I.  COCCACE^E. — Spherical   forms    only ;     division 

occurs  in  one  or  more  directions. 

Genus  1.  MICROCOCCUS  (Staphylococcus). — Division 
in  one  direction  only,  but  irregular,  so  that  the 
cocci  after  division  form  irregular  clusters. 

Genus  2.  STREPTOCOCCUS. — Division  in  one  plane, 
but  regular,  so  that  the  cocci  form  chains. 

Genus  3.  MERISMOPEDIA  (Tetracoccus). — Division 
in  two  directions  at  right  angles  to  each  other, 
1  See  Dobell,  Journ.  of  Genetics,  if,  pp.  201,  325, 


CLASSIFICATION  OF  BACTERIA  17 

but  in  the  same  plane,  so  that  lamellae  or  plates 
are  formed. 

Genus  4.  SAKCINA. — -Division  in  three  directions  at 
right  angles  to  one  another  and  in  two  planes,  eo 
that  cubical  masses  are  formed. 

Genus  5.     Ascococcus. — Cocci  which  develop  in  a 

gelatinous  matrix. 
Family  II.     BACTERIACE^E. — Rods,  straight  or  curved,  at 

some  period  of  the  life-history,  though  coccoid  and 

other  forms  may  occur. 

Genus  1.  BACTERIUM. — Straight  rods  ;  endospore 
formation  does  not  occur. 

Genus  2.  BACILLUS. — Straight  rods  ;  endospore 
formation  occurs. 

Genus  3.  LEUCONOSTOC. — Cocci  and  rods  ;  arthro- 
spore  formation  occurs  in  the  coccoid  forms. 

Genus  4.  CLOSTRLDIUM. — The  same  as  bacillus,  but 
the  spore-bearing  rods  are  enlarged  and  club- 
shaped. 

Genus  5.  SPIRILLUM. — Spiral  rods  ;  spore  forma- 
tion does  not  occur. 

Genus  6.     VIBRIO. — Spiral  rods  ;    spore  formation 

occurs . 
Family    III.     LEPTOTRICHE^J. — These    are    unbranching 

thread  forms. 
Family    IV.     CLADOTRICHE^:. — These    are    thread    forms 

showing  true  but  not  dichotomous  branching. 

There  are  many  features  in  this  classification  which 
are  of  practical  value.  The  distinction  made  between  a 
bacterium  and  a  bacillus,  for  example,  is  convenient. 
Formerly  a  short  rod  was  termed  a  bacterium,  and  a  long 
rod  a  bacillus,  but  such  a  division  is  an  arbitrary  one,  and 
at  one  stage  of  its  life-history  an  organism  might  be  a  bac- 
terium and  at  another  a  bacillus.  The  term  "  bacterium  " 

2, 


18  A  MANUAL  OF  BACTERIOLOGY 

is  now  but  little  used  in  this  sense,  and  any  straight  rod 
is  termed  a  bacillus.  The  term  "  staphylococcus  "  is 
one  frequently  met  with  ;  it  is  practically  synonymous 
with  micrococcus,  and  refers  to  cocci  which  are  aggregated 
into  groups  or  clusters.  Of  the  twisted  rods,  a  simple 
curved  rod  is  now  known  as  a  vibrio,  a  definitely  cork- 
screw form  of  three  or  a  few  turns  is  a  spirillum,  a  long  and 
flexible  twisted  filament  is  a  spirochaeta.  The  systematic 
position  of  the  Spirochaetse  has  given  rise  to  controversy. 
The  parasitic  ones  (e.g.  that  of  relapsing  fever)  are  com- 
monly regarded  as  Protozoa,  but  Dobell1  dissents  from 
this  view  and  considers  them  all  to  be  much  more  closely 
allied  to  the  Bacteria,  which  he  classifies  as  follows  : 

rTrichobacteria     (£occ°id*a 

{itaL^saa: 

v  SPIRO  CHAETOIDEA 


\Cristispira 
Saprospira 


Another  classification  is  that  proposed  by  Migula.2 
The  Bacteria  are  divided  into  two  orders  :  the  Eubacteria 
— bacteria  proper — the  cells  of  which  contain  neither 
sulphur  granules  nor  a  colouring  matter,  bacterio-purpurin  ; 
and  the  Thiobacteria,  the  cells  of  which  contain  sulphur 
granules  and  may  be  coloured  with  bacterio-purpurin. 
The  Eubacteria  are  divided  into  five  families  :  (1)  Coccacese, 
(2)  Bacteriaceae,  (3)  Spirillacea3,  (4)  Chlamydo-bacteriaceae, 
and  (5)  Beggiatoacea3.  These,  again,  are  subdivided  into 
many  genera,  based  partly  on  the  mode  of  division  and 
partly  on  the  number  and  on  the  arrangement  of  the 
flagella  upon  the  organisms.  The  Coccacese — globular  cells 
— contain  the  genera  Streptococcus,  Micrococcus,  Sarcina 
(non-motile),  and  Planococcus  and  Planosarcina  (motile) ; 
the  Bacteriaceae  are  defined  as  long  or  short  cylindrical 

1  Proc.  Roy.  Soc.  Lond.,  B,  vol.  85,  1912,  p.  186. 

2  System  der  Bakterien,  1897. 


CONDITIONS  OF  LIFE        ,  19 

rods,  straight  and  never  spiral ;  division  in  one  direction 
only  after  elongation  of  the  rods  ;  and  this  family  has 
three  genera  :  (a)  Bacterium — non- flagellated  cells,  often 
with  endospore  formation  ;  (b)  Bacillus — cells  possessing 
both  lateral  and  polar  flagella,  often  with  endospore  forma- 
tion ;  (c)  Pseudomonas — cells  with  polar  flagella  only, 
rarely  endospore  formation.  The  Spirillacese  are  curved 
or  spiral  rods,  and  include  (a)  Spirosoma,  non-motile 
forms,  (6)  Microspira,  motile  forms  with  one  polar  flagellum, 
(c)  Spirillum,  motile  forms  with  two  or  more  polar  flagella. 
Various  pointed  organisms  have  been  described  as 
"  fusiform*  Bacteria,"  e.g.  in  Vincent's  angina  (see  Chap. 
VIII),  but  Dobell  expresses  the  opinion  that  these  more 
probably  belong  to  the  Fungi. 

The  nomenclature  of  bacterial  species  is  at  present  in  a  chaotic 
condition.  In  botanical  and  zoological  nomenclature  every  species 
has  a  binomial  name,  the  first  being  the  generic,  the  second  the 
specific  name.  Many  bacterial  species  have  received  trinomial  or 
multinomial  names,  which  should  be  inadmissible.  The  specific 
name  first  given  to  an  organism  must  stand  unless  it  has  been  used 
for  some  other  species. 

Conditions  of  Life  of  Bacteria 

Bacteria,  being  living  organisms,  must  be  supplied  with 
suitable  nutritive  substances  in  order  that  their  life- 
processes — nutrition,  reproduction,  and  the  like — may  be 
carried  on  in  a  normal  manner.  Being  devoid  of  chloro- 
phyll they  are  mainly  dependent  upon  complex  organic 
compounds  for  the  carbon,  hydrogen,  and  nitrogen  which 
enter  into  their  composition,  these  elements  being  derived 
for  the  most  part  from  proteins  and  carbohydrates.  Some 
bacteria,  however,  are  able  to  obtain  the  requisite  nitrogen 
from  such  comparatively  simple  compounds  as  ammonia, 
ammonium  carbonate,  or  nitrates,  and  one  group  can 
make  direct  use  of  the  atmospheric  nitrogen.  Certain 


20  A  MANUAL  OF  BACTERIOLOGY 

inorganic  salts,  sulphates,  phosphates,  and  sodium  chloride, 
also  seem  to  be  necessary  for  normal  development.  These 
nutrient  substances  must  be  presented  to  the  bacteria  in 
association  with  water,  for  without  water  bacterial  activity 
ceases,  though  in  the  dry  state  many  forms,  and  especially 
their  spores,  may  retain  their  vitality  for  a  considerable 
time  ;  absolute  desiccation,  however,  is  rapidly  fatal  to 
many. 

Temperature  is  also  an  important  factor.  Though  the 
growth  of  many  species  occurs  through  a  wide  range, 
there  is  for  almost  all  an  optimum  at  which  growth  is  best, 
and  of  a  range  not  exceeding  5°  or  10°.  Growth  usually 
ceases  below  10°  C.,  but  cold  does  not  destroy  bacterial 
life  ;  after  exposure  to  the  intense  cold  produced  by  the 
evaporation  of  liquid  oxygen  (—  170°  C.)  for  weeks,  or  of 
liquid  hydrogen  (—  252°  C.)  for  ten  hours,  bacteria  and 
their  spores  will  grow  and  germinate,  and  their  chromo- 
genic  and  pathogenic  properties  seem  to  be  unaltered.1 
On  the  other  hand,  bacterial  growth  usually  ceases  when 
the  temperature  exceeds  40°  C.  or  thereabouts,  and  most 
bacteria  without  spores  are  destroyed  within  half  an  hour 
by  a  temperature  of  65°  C.  The  spores  are  far  more 
resistant  ;  some  may  even  be  boiled  for  a  short  time 
without  losing  their  vitality,  but  prolonged  boiling  is  fatal 
to  both  bacteria  and  their  spores.  There  is,  however, 
a  group  of  so-called  thermophilic  bacteria,  which  thrive 
best  at  a  temperature  of  60°  to  70°  C.  They  occur  in  the 
soil  and  in  water,  and  are  probably  of  considerable  import- 
ance in  the  natural  fermentations  accompanied  by  the 
evolution  of  heat,  such  as  are  met  with  in  manure  heaps, 
the  heating  of  hay,  and  firing  of  moist  cotton.2 

Free  oxygen  is  essential  to  the  growth  of  some  organisms  ; 

1  Macfadyen  and  Rowland,  Proc.  Roy.  Soc.  Lond.,  1900. 

2  Macfadyen  and  Blaxall,  Journ.  of  Path,  and  BacL,  November  1894. 
and  Trans.  Jenner  Inst.  of  Prev.  Med.,  vol.  ii,  1899,  p.  162. 


BACTERIAL  DEVELOPMENT  21 

these  are  termed  strictly  aerobic.  Others  will  not  develop 
in  its  presence,  strictly  anaerobic  ;  others,  again,  while 
preferably  aerobic  or  anaerobic,  will  grow  in  the  absence, 
or  in  the  presence,  of  oxygen,  and  are  respectively  termed 
facultative  anaerobic  or  facultative  aerobic.  Some 
organisms  are  strictly  parasitic  on  animals  or  plants  ; 
others  live  in  water,  soil,  decaying  matter,  etc. — these  are 
termed  saprophytes  ;  and  many  are  able  to  exist  either  as 
parasites  or  as  saprophytes. 

Bacterial  development  is  much  influenced  by  the  presence 
of  foreign  substances  in  the  nutrient  medium.  A  number 
of  metallic  and  other  salts,  chlorine,  bromine,  and  iodine, 
carbolic  acid,  salicylic  acid,  etc.,  have  an  injurious  effect 
upon  bacterial  life,  inhibiting  or  stopping  growth,  or 
killing  the  organisms  outright ;  these  are  of  considerable 
practical  importance  and  are  known  as  germicides,  anti- 
septics, and  disinfectants.  The  products  produced  in  the 
nutrient  medium  by  the  bacteria  themselves  also  sooner 
or  later  inhibit  or  stop  further  growth  ;  a  familiar  instance 
of  this  is  seen  in  the  alcoholic  fermentation  of  sugar  by 
yeast,  which  ceases  when  the  amount  of  alcohol  reaches 
12  to  14  per  cent.  The  same  reason  probably  accounts 
for  the  fact  that  growths  of  bacteria  in  culture  tubes 
frequently  do  not  spread  all  over  the  surface  of  the  nutrient 
medium,  and  why  our  cultures  sometimes  die  out  more 
rapidly  than  might  be  expected. 

Another  point  affecting  bacterial  life  is  the  presence  of 
a  mixture  of  organisms  in  the  same  nutrient  medium.  If 
there  be  a  very  vigorous  form,  it  may  ultimately  grow 
and  multiply  to  such  an  extent  as  to  crowd  out  and  finally 
kill  the  other  forms  with  which  it  is  associated,  and  if  the 
nutrient  medium  equally  favour  two  species,  that  one 
which  is  in  an  excess  at  the  beginning  may  outgrow  the 
other.  The  occurrence  of  what  has  been  termed  symbiosis 
is  of  considerable  interest  in  the  life  of  micro-organisms, 


22  A  MANUAL  OF  BACTERIOLOGY 

and  too  little  attention  has  hitherto  been  paid  to  it.  This 
is  the  co-existence  of  two  or  more  species  which  together 
bring  about  certain  changes.  For  example,  in  the  well- 
known  ginger-beer  plant,  Marshall  Ward1  isolated  several 
yeasts,  bacteria,  and  moulds  ;  of  these,  one  of  the  yeasts 
and  one  of  the  bacteria  together  induce  the  particular 
changes  in  a  saccharine  fluid  to  which  ginger  has  been 
added,  which  render  the  mixture  like  ginger-beer,  and 
these  changes  do  not  occur  unless  both  species  develop 
together. 

Another  extraordinary  feature  exhibited  by  bacteria  is 
the  selective  action  exerted  on  certain  substances  which 
contain  isomerides  or  right-  and  left-handed  modifications 
of  a  substance.  The  Bacillus  eihaceticus  attacks  mannitol 
but  not  dulcitol,  two  alcohols  which  are  very  similar  in 
taste  and  properties  and  possess  the  same  simple  chemical 
formula. 

By  a  series  of  most  brilliant  researches  Emil  Fischer 
succeeded  in  determining  the  constitution  of  the  various 
sugars,  and,  what  is  more,  has  produced  them  artificially 
in  the  laboratory.  The  natural  sugars  are  all  compounds 
with  dissymmetric  molecules,  powerfully  affecting  the 
beam  of  polarised  light,  but  when  prepared  artificially 
they  are  without  action  on  polarised  light,  because  the 
artificial  product  consists  of  equal  numbers  of  left-handed 
and  right-handed  molecules,  and  the  molecules  of  the  one 
neutralise  the  molecules  of  the  other,  thus  giving  rise  to 
a  mixture  which  does  not  affect  the  polarised  beam. 

By  the  action  of  micro-organisms,  however,  on  such  an 
inactive  mixture  the  one  set  of  molecules  is  sought  out  by 
the  microbes  and  decomposed,  leaving  the  other  set  of 
molecules  untouched,  and  the  latter  now  exhibit  their 
specific  action  on  polarised  light,  an  active  sugar  being 
thus  obtained. 

1  Phil.  Trans.  Roy.  Soc.  Lond.,  vol.  clxxxiii,  1892,  p.  125. 


EFFECT  OF  PHYSICAL  AGENTS      23 

Fructose  was  one  of  the  principal  artificial  sugars  pre- 
pared by  Fischer  ;  it  is  inactive,  but  consists  of  an  equal 
number  of  molecules  of  oppositely  active  sugars  termed 
"  Isevulose."  One  set  of  these  Isevulose  molecules  turns 
the  plane  of  polarisation  to  the  right,  another  set  to  the 
left — right-  and  left-handed  Isevulose.  The  left-handed 
Isevulose  occurs  in  nature,  while  the  right-handed  Isevulose, 
so  far  as  is  known,  does  not. 

Now,  on  putting  brewer's  yeast  into  a  solution  of 
fructose,  the  inactive  artificial  product,  the  yeast  organisms 
attack  the  left-handed  Isevulose  molecules  and  convert 
them  into  alcohol  and  C02,  while  the  right-handed  Isevulose 
is  left  untouched. 

Pressure,  unless  very  great,  has  little  effect  on  bacteria. 
Roger  investigated  the  effects  of  high  pressure  on  certain 
organisms  in  bouillon  cultures.  Pressures  of  200  to 
250  kilos,  per  square  centimetre  had  no  effect  ;  by  raising 
the  pressure  to  3000  kilos,  per  square  centimetre  one- 
third  of  streptococci  were  killed,  and  of  anthrax  without 
spores  a  good  many  ;  while  sporing  anthrax,  Micrococcus 
pyogenes,  var.  aureus,  and  the  colon  bacillus  were  un- 
affected.1 

Our  countrymen  Downes  and  Blunt  first  called  attention 
to  the  injurious  effect  of  light  upon  bacteria.  If  plate 
cultures  be  prepared  and  exposed  to  sunlight,  a  portion 
of  the  plate  being  protected  from  its  action,  as  by  sticking 
on  a  letter  cut  out  of  black  paper,  and  the  preparation 
afterwards  incubated,  it  will  be  found  that  the  colonies 
develop  at  the  protected  portion  only,  those  parts  which 
have  been  exposed  to  sunlight  remaining  sterile.  Although 
this  action  of  sunlight  may  occasionally  be  due  to  chemical 
changes  in  the  medium,  resulting  in  the  production  of 

1  Bacteria  being  so  minute,  the  actual  pressure  on  a  bacterial  cell, 
even  with  these  high  pressures,  is  small.  If,  for  example,  a  bacterium 
measures  1  p.  by  5  p.,  a  pressure  of  1000  kgrm.  per  square  centimetre 
\vould  be  but  0-05  grin,  (f-  grain)  on  the  cell. 


24  A  MANUAL  OF  BACTERIOLOGY 

ozone  or  other  germicidal  bodies,  the  experiments  of 
Marshall  Ward  and  others  have  conclusively  shown  that 
germicidal  action  may  be  caused  by  the  direct  action  of 
the  light,  the  violet  and  ultra-violet  rays  being  those 
concerned,  and  the  red  end  of  the  spectrum  has  no  effect. 
Ultra-violet  rays  may  produce  mutations  of  the  anthrax 
bacillus  (see  p.  6).  The  Rontgen  rays  seem  to  have  little 
or  no  influence  upon  bacteria,  but  the  results  that  have 
been  obtained  are  somewhat  contradictory. 

The  radium  emanations  with  prolonged  exposure  and 
near  contact  are  germicidal  to  non-sporing  organisms.1 

Electricity,  per  se,  has  also  usually  little  effect.  When 
the  current  is  passed  directly  through  the  cultures  electro- 
lysis takes  place,  and  the  products  formed  may  destroy 
the  bacteria  ;  currents  of  high  potential,  however,  may 
inhibit  growth. 

Living  motile  bacilli  are  very  sensible  to  induced  currents 
of  electricity,  immediately  orientating  themselves  in  the 
direction  of  the  current,  while  dead  or  paralysed  bacilli  are 
unaffected. 

Bacterial  Products 

The  chemical  changes  produced  by  micro-organisms  are 
chiefly  analytic  or  destructive — the  formation  of  simpler 
from  more  complex  bodies.  This  analytic  faculty  is 
present  to  a  marked  degree  in  the  process  known  as 
putrefaction.  Putrefaction  is  a  term  applied  to  the  decom- 
position of  organic,  especially  protein,  matter  after  the 
death  of  the  animal  or  plant.  It  is  usually  accompanied 
by  the  evolution  of  foul-smelling  gases  and  by  solution 
of  the  solid  material.  A  large  number  of  organisms  are 
concerned  in  this  process,  particularly  a  group  to  which 
Hauser  gave  the  name  of  Proteus.  The  first  changes 
which  occur  are  the  formation  of  proteoses  and  peptone, 
1  See  Green,  Proc.  Roy.  Soc.  Lond.,  vol.  Ixxiii,  1904,  p.  375. 


INDOLE  25 

then  leucin,  tyrosin,  and  glycocol,  and  basic  compounds 
to  which  the  name  of  ptomine  has  been  given  ;  next 
indole,  skatole,  and  phenol,  and  volatile  fatty  acids  ;  and 
lastly,  mercaptans,  sulphuretted  hydrogen,  marsh  gas, 
ammonia,  carbonic  acid,  and  hydrogen. 

In  view  of  its  practical  importance  in  bacteriological 
analysis  and  the  identification  of  species,  indole  may  here 
be  referred  to  at  some  length. 

Indole. — Indole  (C8H7N)  is  a  product  of  the  putrefactive 
decomposition  of  proteins  containing  a  tryptophane 
nucleus  and  is  formed  during  the  growth  of  many  organisms, 
and,  since  one  species  may  produce  it  and  another  allied 
one  may  not,  the  determination  of  its  presence  or  absence 
in  the  culture  may  be  of  value  in  the  identification  of 
organisms.  The  detection  of  indole  is  based  on  the 
reaction  with  nitrous  acid,  with  which  it  gives  a  fine 
purplish-red  coloration.  In  order  to  test  for  it,  the 
organism  is  grown  in  a  fluid  medium  for  twenty-four  to 
forty-eight  hours  or  longer,  1  c.c.  of  a  0-1  per  cent,  solution 
of  sodium  nitrite  is  added  to  every  10  c.c.  of  the  culture, 
and  a  few  drops  of  pure  concentrated  sulphuric  acid  or 
of  hydrochloric  acid  are  allowed  to  trickle  slowly  down  the 
side  of  the  test-tube,  which  is  inclined  with  its  mouth 
away  from  the  operator.  As  the  acid  runs  down,  it  is 
mixed  with  the  fluid  ;  a  colour  varying  from  pale  pink  to 
pale  purple  indicates  the  presence  of  indole.  A  control 
tube,  uninoculated,  should  also  be  similarly  tested  to  make 
sure  that  the  reaction  is  due  to  the  products  of  the  growth 
of  the  organism.  The  culture  fluid  usually  employed  is 
peptone  water,  preferably  2  per  cent.,  but  some  samples 
of  "  peptone  "  occasionally  fail  to  give  the  indole  reaction 
when  organisms  are  grown  in  media  prepared  from  them  ; 
the  right  kind  of  peptone  must,  therefore,  be  used.  As 
the  dilute  solution  of  sodium  nitrite  is  unstable,  a  stock 
5  per  cent,  solution  may  be  kept ;  2  c.c.  of  this  solution 


26  A  MANUAL  OF  BACTERIOLOGY 

are  diluted  to  100  c.c.  with  distilled  water  at  the  time  of 
making  the  test,  and  1  c.c.  of  this  dilution  is  added  to 
every  10  c.c.  of  the  culture.  The  addition  of  the  acid 
liberates  free  nitrous  acid,  which  reacts  with  any  indole 
present,  and  yields  a  pink  colour.  Sometimes  when  the 
reaction  is  apparently  absent  or  feeble,  it  may  be  obtained 
or  intensified  by  placing  the  tube  in  the  blood-heat 
incubator  for  half  an  hour.  The  sulphuric  acid  should  be 
pure  and  free  from  oxides  of  nitrogen,  hence  hydrochloric 
acid  is  often  preferable. 

A  more  delicate  method  of  testing  is  to  run  a  little 
hydrochloric  acid  down  the  side  of  the  tube,  so  that  a  layer 
forms  at  the  bottom,  the  nitrite  having  been  previously 
added  to  the  culture  if  required.  A  pink  ring  at  the 
juncture  of  the  hydrochloric  acid  and  culture  indicates 
the  presence  of  indole.  The  pink  pigment,  the  product 
of  the  reaction,  may  be  extracted  by  shaking  with  a  little 
amylic  alcohol. 

Other  delicate  and  more  certain  reagents  for  the  detection  of 
indole  are  para-dimethylamidobenzaldehyde  (14  grm.,  dissolved  in 
absolute  alcohol  380  c.c.,  hydrochloric  acid  80  c.c.).  To  about 
10  c.c.  of  culture  5  c.c.  of  this  solution  are  added,  and  then  5  c.c. 
of  a  saturated  aqueous  solution  of  potassium  persulphate  ;  indole 
gives  a  pink  or  red  colour.  Another  test  is  /3-naphthaquinone- 
sodium-mono-sulphonate  (2  per  cent,  aqueous  solution),  which  gives, 
when  the  mixture  is  rendered  alkaline  with  caustic  potash,  a  blue 
or  blue -green  colour  or  precipitate.  The  coloured  compound  may 
be  extracted  with  chloroform,  in  which  it  yields  a  red  solution. 

Peptone  water  is  by  no  means  a  good  culture  medium, 
and  broth  may  therefore  be  employed,  but  it  should  be 
free  from  dextrose.  Peptone  water  with  the  addition  of 
a  little  rabbit's  serum  is  perhaps  the  best  culture  medium 
for  the  production  of  indole. 

The  presence  of  dextrose,  saccharose,  glycerin,  or  lactose 
in  quantity  exceeding  about  0-25  per  cent,  prevents  the 
formation  of  indole  in  broth  by  bacteria.  Broth  prepared 


INDOLE  REACTION  27 

in  the  ordinary  way  usually  contains  a  little  dextrose 
derived  from  the  glycogen  in  the  meat,  and  this  probably 
explains  why  the  indole  reaction  is  generally  much  more 
marked  in  a  peptone  water  than  in  a  broth  culture,  although 
the  latter  is  a  better  nutrient  soil.  In  order  to  prepare  a 
soil  free  from  dextrose,  the  acid  beef  broth  used  in  the 
preparation  of  nutrient  broth  should  be  inoculated  with 
the  colon  bacillus  and  incubated  for  twenty-four  hours, 
and  the  peptone  beef  broth  prepared  from  it.  The 
dextrose  is  consumed  and  no  indole  is  formed.1 

Some  bacteria  not  only  form  indole  but  also  produce 
nitrites  in  the  culture  medium  by  the  reduction  of  the 
nitrates  present  in  the  peptone,  etc.,  used  in  making  the 
nutrient  medium,  in  which  case  the  addition  of  pure 
sulphuric  or  hydrochloric  acid  alone  suffices  to  bring 
out  the  pink  indole  reaction.  This  forms,  therefore,  an 
additional  means  of  distinguishing  organisms,  and  is 
employed  especially  for  the  recognition  of  the  cholera 
spirillum,  which,  if  grown  in  peptone  water,  gives  the 
indole  reaction  (or,  as  it  has  been  termed,  "  the  cholera 
red  reaction  ")  on  the  addition  of  acid  alone.  The  reaction 
can  be  obtained  as  early  as  twelve  hours  after  inoculation, 
and  becomes  very  marked  in  twenty-four  to  forty-eight 
hours. 

If  indole  is  formed  only  in  small  quantities,  100  c.c. 
of  the  culture  may  be  distilled  ;  the  first  20  c.c.  of  the 
distillate  will  contain  the  bulk  of  the  indole. 

This  "  indole -reaction  "  is  not  necessarily  always  due 
to  indole  ;  the  writer  has  shown2  that  the  indole-like 
reaction  obtained  with  cultures  of  the  diphtheria  and 
pseudo-diphtheria  bacilli  is  owing  to  the  presence  of 
skatole-carboxylic  acid.  This  substance  is  distinguished 
from  indole  by  being  non- volatile.  To  make  sure  of  the 

1  T.  Smith,  Journ.  of  Exper.  Med.,  vol.  ii,  1897,  p.  543. 

2  Trans.  Path.  Soc.  Lond.,  vol.  lii,  pt.  ii,  1901,  p.  113. 


28  A  MANUAL  OF  BACTERIOLOGY 

presence  of  indole,  the  culture  should  therefore  be  made 
alkaline  with  caustic  soda  and  distilled. 

Skatole  (methyl  indole)  seems  also  to  be  formed  by  some  organisms. 
It  is  volatile  like  indole,  but  if  a  solution  containing  it  be  boiled 
with  an  acid  solution  of  dimethylamidobenzaldehyde  (5  per  cent. 
in  10  per  cent,  sulphuric  acid)  it  yields  a  blue  colour,  which  gives  a 
blue  solution  in  chloroform. 

Nitrification. — Another  important  series  of  changes  is 
that  included  under  the  term  "  nitrification."  As  men- 
tioned before,  protein,  albuminoid,  and  other  complex 
nitrogenous  matters  and  urea,  all  of  which  are  valuable 
manures  for  plant  life,  cease  to  be  so  unless  bacteria  are 
present. 

It  is  necessary,  in  fact,  for  the  nitrogenous  matter  to 
be  converted  into  nitrates,  in  which  form  alone  is  it  avail- 
able for  the  nutrition  of  plants. 

Although  so  important,  extremely  small  quantities  of 
nitrates  are  present  in  the  soil ;  in  fertile  soils,  for  example, 
under  some  conditions  there  may  be  as  little  as  one  part 
of  nitrogen  in  1,000,000,  and  there  is  often  less  than  ten 
parts.  The  bodies  yielding  nitric  acid  in  the  soil  are  : 
(1)  free  nitrogen  ;  (2)  small  quantities  of  nitrates  in  rain- 
water ;  (3)  ammonium  salts,  applied  intentionally  or 
carried  to  the  soil  by  rain  or  derived  from  the  decay  of 
organic  matter ;  (4)  various  nitrogenous  organic  sub- 
stances arising  from  the  decay  of  animal  and  vegetable 
matters. 

With  regard  to  the  production  of  nitric  acid  from 
nitrogenous  organic  matters  very  little  was  formerly 
known.  In  1877  Schloesing  and  Miintz  by  an  ingenious 
experiment  showed  that  nitrification  (as  the  production 
of  nitric  acid  is  termed)  of  nitrogenous  organic  matter  is 
brought  about  by  living  organisms  in  the  soil.  Sewage 
was  passed  continuously  through  a  tube  containing  a 
mixture  of  ignited  quartz  sand  and  limestone.  After 


NITRIFICATION  29 

three  weeks  nitrates  began  to  appear  in  the  effluent  and 
increased  to  such  an  extent  that  finally  the  filtered  sewage 
contained  no  ammonia.  After  this  had  continued  for 
some  weeks  chloroform  vapour  was  passed  at  the  same 
time  through  the  tube,  with  the  result  that  in  ten  days 
after  the  introduction  of  the  chloroform  all  nitrates  dis- 
appeared from  the  effluent. 

Subsequently  the  passage  of  chloroform  vapour  was 
discontinued,  but  nitrification  did  not  resume  until  the 
washings  from  10  grm.  of  garden  soil  were  added.  Eight 
days  after  this  addition  nitrates  again  appeared  in  the 
effluent  (this  was  confirmed  by  Warington).  Evidently 
the  chloroform  vapour  acted  as  an  antiseptic  and  killed 
the  nitrifying  organisms,  while  the  addition  of  soil  washings 
re-inoculated  the  material. 

Shortly  after  this  Schloesing  and  Miintz  found  that 
exposure  of  soil  to  100°  C.  for  an  hour  destroyed  the 
power  of  mducing  nitrification.  Soils  thus  treated  were 
exposed  to  a  current  of  air,  purified  by  ignition,  without 
nitrification  taking  place  ;  the  addition  of  a  little  un- 
heated  mould  was,  however,  sufficient  to  cause  nitrifica- 
tion to  recommence.  They  also  tried  seeding  the  sterilised 
soils  with  various  Hyphomycetes,  etc.,  without  result. 

In  1884  Warington  concluded  that  the  factor  deter- 
mining the  formation  sometimes  of  nitric  acid  and  some- 
times of  nitrous  acid  was  a  difference  in  the  character  of 
the  organisms  ;  for  it  is  possible  to  have  two  similar 
solutions  under  identical  conditions,  and  for  nitrites  to  be 
produced  in  the  one,  and  nitrates  in  the  other. 

In  1886  Munro  showed  that  the  process  of  nitrification 
could  take  place  in  solutions  practically  destitute  of 
organic  matter. 

Nitrification  in  the  soil  takes  place  in  three  stages  : 

I.  Ammonisation. — When  complex  organic  compounds 
such  as  albuminoids  are  applied  to  the  land  they  are 


30  A  MANUAL  OF  BACTERIOLOGY 

broken  up ;  first  they  become  liquefied,  peptone-like 
bodies  being  produced  ;  these  are  then  further  acted  upon 
and  we  get  alkaloidal  substances  in  small  quantity,  indole, 
skatole,  leucin,  and  tyrosin  and  amino-acids,  valerianic 
acid,  volatile  fatty  acids,  lactic  acid,  etc. 

These  changes  are  brought  about  by  numbers  of 
organisms,  among  which  the  varieties  of  Proteus  (formerly 
known  as  Bacterium  termo),  B.  mesentericus,  B.  mycoides, 
B.  fluorescens  liquefaciens,  and  B.  putrificus  are  the  more 
important. 

The  nitrogenous  compounds  are  then  further  acted  upon 
and  ammonium  salts  are  formed.  According  to  Emile 
Marchal,  ammonisation  takes  place  essentially  under  the 
influence  of  microbes  living  in  the  upper  layers  of  the  soil. 
The  Bacillus  mycoides  is  one  of  the  most  energetic  of  these, 
and  seems  to  play  a  double  role,  being  ammonising  in  the 
presence  both  of  nitrogenous  organic  substances  and  of 
nitrates.  Urea  is  ammonised  especially  by  the  Micro- 
coccus  urece. 

II.  Nitrosation. — The  ammoniacal  salts   are   next  con- 
verted into  nitrites.     The  nitrous  organisms  can  probably 
attack  nitrogenous  organic  substances  such  as  asparagine 
and  milk,  but  only  feebly,  milk  being  much  more  rapidly 
nitrified  when  the  nitrous  organisms  are  mixed  with  other 
species.     The  organisms  bringing  about  this  change  are 
short,   stumpy,   motile   bacilli   with   single   polar   flagella 
which  are  grouped  under  the   generic   name  of  Pseudo- 
monas. 

III.  Nitratation. — These    nitrites    are    then    converted 
into  nitrates.     The  "  nitric  "  organisms  are  minute  non- 
motile  bacilli  known  as  Nitrobacter. 

Stages  II  and  III  are  brought  about  by  different  species, 
the  nitric  organisms  having  no  effect  whatever  on  ammonia, 
but  acting  only  after  this  has  been  oxidised  into  nitrous 
acid  by  the  nitrous  forms. 


NITRIFICATION  31 

The  discovery  of  Dr.  Munro  that  organisms  will  grow  in  purely 
inorganic  solutions  has  been  made  use  of  for  the  isolation  of  the 
different  species.  Solutions  such  as  the  following  have  been  used  : 

For  the  Nitrous  Organisms.  For  the  Nitric  Organisms. 

Ammonium  chloride,  0-5  grm.  Potassium  nitrite,  0-3  grin. 

Potassium  phosphate,  0-1  grm.  Potassium  phosphate,  0-1  grm. 

Magnesium  sulphate,  0-02  grm.  Magnesium  sulphate,  0-05  grm. 

Calcium  chloride,  0-01  grm.  Calcium  carbonate,  5  grm. 

Calcium  carbonate,  5  grm.  Distilled  water,  1000  c.c. 
Distilled  water,  1000  c.c. 

These  are  seeded  with  traces  of  earth,  and  by  carrying  on  the 
cultivation  for  many  generations  a  large  number  of  organisms  are 
eliminated.  This  method  does  not  lead  to  a  pure  cultivation,  for 
several  forms  besides  the  nitrifying  organisms  persistently  maintain 
themselves  in  these  mineral  solutions. 

So  recourse  was  had  to  gelatin  plate  cultivations.  Although 
several  organisms  were  isolated  in  this  manner,  none  of  them 
possessed  the  slightest  nitrifying  power. 

Frankland,  and  later  Warington  (1890),  succeeded  in  isolating 
nitrous  organisms  by  the  dilution  method.  Nitrifying  solutions 
were  diluted,  and  traces  inoculated  into  ammoniacal  solutions  ;  in 
some  of  these  nitrification  occurred,  although  no  growth  could  be 
obtained  on  gelatin,  and  they  were  found  to  contain  the  nitrous 
organism  only.  A  little  later  Winogradsky  isolated  nitrous 
organisms,  first  by  modified  gelatin  plates,  and  afterwards  by  the 
silica  jelly  method. 

This  is  carried  out  as  follows  :  Sodium  carbonate  is  fused  in  the 
blowpipe,  and  fine  white  sand  is  added  so  long  as  effervescence  is 
produced.  The  mass  is  allowed  to  cool,  and  is  then  dissolved  in 
water.  The  solution  is  poured  into  an  excess  of  very  dilute  hydro- 
chloric acid  (silicic  acid  and  sodium  chloride  being  formed).  The 
solution  is  dialysed  and  sterilised.  For  use,  some  of  this  is  placed 
in  a  sterile  dish  and  is  mixed  with  the  following  solution  and 
inoculated  : 

Ammonium  sulphate        ....  0-4  grm. 

Magnesium  sulphate        .          .          .  0-5     ,, 

Di-potassium  hydrogen  phosphate    .          .  0-1     ,, 

Calcium  chloride     .....  trace 

Sodium  carbonate  .....  0-6-0-9  grin. 

Water 100  c.c. 

This  mixture  sets  to  a  jelly  in  five  to  fifteen  minutes. 
Winogradsky  has  also  made  use  of  agar  for  plates,   but   this 


32  A  MANUAL  OF  BACTERIOLOGY 

medium  is  not  so  suitable  as  the  silica  jelly.  A  2  per  cent,  aqueous 
agar  is  prepared  and  poured  into  Petri  dishes  ;  the  film  is  then  sown 
with  Proteus,  and  allowed  to  grow  for  seven  to  ten  days.  It  is 
then  thoroughly  washed,  collected,  melted,  and  mixed  with  the  salts 
mentioned  above.  The  object  of  growing  the  Proteus  upon  it  as  a 
preliminary  is  to  eliminate  the  organic  matter  admixed  with  the  agar. 

Nitrification  in  the  soil  is  thus  brought  about  by  two 
groups  of  organisms.  The  first  oxidises  ammonia  into 
nitrous  acid,  and  is  isolated  by  successive  cultivation  in 
solutions  of  ammonium  carbonate.  The  second  group 
oxidises  nitrous  acid  into  nitric  acid,  and  may  be  separated 
by  successive  cultivations  in  a  solution  of  potassium 
nitrite  containing  a  little  sodium  bicarbonate.  In  the 
soil  the  nitric  and  nitrous  organisms  are  equally  active. 

Besides  the  derivation  of  nitrogen  from  nitrogenous 
compounds,  the  free  atmospheric  nitrogen  is  also  "  fixed  " 
through  the  agency  of  certain  micro-organisms  and 
rendered  available  for  plant  life. 

Thus,  the  Leguminosae  are  able  to  obtain  their  nitrogen 
directly  from  the  nitrogen  of  the  air.  If  the  roots  of  a 
pea,  bean,  or  vetch  be  examined,  numerous  little  nodules 
will  be  found  upon  them ;  these  contain  minute  irregular 
and  Y-shaped  bodies,  which  have  been  termed  "  bac- 
teroids,"  and  seem  to  be  of  the  nature  of  involution 
forms.  On  inoculation  into  suitable  culture  media1  the 
bacteroids  give  rise  to  a  growth  of  a  motile  bacillus  known 
as  Pseudomonas  radicicola  ;  this  "  fixes  "  the  atmospheric 
nitrogen.  The  organisms  penetrate  the  young  roots 
through,  the  root-hairs,  multiply  and  form  a  filamentous 
zooglcea,  which  grows  into  the  tissue  of  the  root  and 
penetrates  the  cells.  Large  amounts  of  nitrogen  are  taken 
up  by  the  bacteroids,  and  are  converted  into  nitrogenous 

1  Such  as  wood-ashes  maltose  agar.  Boil  8  grm.  of  wood-ashes 
with  500  c.c.  of  water  for  one  minute  ;  filter.  To  400  c.c.  of  this  extract 
add  4  grm.  maltose  and  4  grm.  agar.  Boil  until  dissolved  ;  filter,  tube, 
and  sterilise. 


AZOTOBACTER  33 

compounds  which  can  be  assimilated  by  the  plant.  Legu- 
minous plants  grown  from  sterile  seeds  in  a  sterile  soil 
dwindle  and  die,  but  if  inoculated  with  the  organisms 
derived  from  another  plant  of  the  same  species  growth 
becomes  vigorous  ;  if  inoculated  with  those  derived  from 
another  species  growth  still  takes  place,  but  not  nearly  to 
the  same  extent.  The  Leguminosse  thus  store  up  one  of 
the  most  important  elements  of  plant  food,  and  hence 
their  value  in  the  rotation  of  crops.  There  is  apparently 
no  increase  of  nitrogen  compounds  in  the  soil,  the  excess 
found  being  due  to  the  root  residues  remaining.  A  sub- 
stance termed  "  nitragin,"  consisting  of  a  culture  of  these 
root  organisms,  has  been  prepared  as  a  fertiliser.  Nobbe's 
"  nitragin  "  did  not  prove  a  success,  apparently  because 
the  organisms  soon  lose  their  vitality.  A  better  prepara- 
tion, "  nitro-bacterine,"  was  devised  by  Moore  of  the 
United  States  Department  of  Agriculture.  Besides  the 
leguminous  organisms,  other  bacteria  are  present  in  the 
surface  layers  of  the  soil  which  fix  atmospheric  nitrogen. 
The  principal  of  these  are  ovoid  organisms  known  as 
Azotobacter.  This  group  can  be  cultivated  in  a  mannite 
medium,  e.g.  di-potassium  phosphate  0-2  grm.,  mannite 
20  grm.,  water  1  litre.  This  may  be  used  for  isolation 
by  converting  into  an  agar  medium  by  the  addition  of 
2  per  cent.  agar.  Prof.  Bottomley  has  succeeded  in 
obtaining  a  powder  preparation  of  Azotobacter,  which 
retains  its  vitality  for  months,  and  the  preparation  properly 
applied  to  poor  soils  produces  astonishing  results. 

It  has  been  found  that  partial  sterilisation  of  the  soil, 
e.g.  by  heat,  increases  its  fertility,  whereas  it  might  have 
been  supposed  that  such  a  procedure  would  decrease  the 
fertility  by  destruction  of  nitrogen-fixers.  Russell  and 
Hutchinson  suggest  that  in  ordinary  soil  amoebae  and  other 
protozoa  devour  and  keep  down  the  bacteria  ;  by  the 
sterilisation  the  protozoa  are  destroyed  and  the  more 

3 


34  A  MANUAL  OF  BACTERIOLOGY 

resistant  bacteria  are  then  free  to  develop.  Greig-Smith,1 
however,  denies  that  phagocytic  protozoa  possess  any 
power  of  limiting  the  number  of  bacteria  in  the  soil,  and 
ascribes  the  effect  of  soil  sterilisation  to  an  action  on  the 
bacterio-toxins  and  nutrients  of  the  soil. 

Besides  nitrifying  bacteria  many  de-nitrifying  organisms 
occur  in  the  soil.  They  may  (1)  reduce  nitrates  to  nitrites  ; 
(2)  remove  oxygen  from  nitrates  and  nitrites  and  form 
ammonia  ;  (3)  form  nitrous  and  nitric  oxides  or  nitrogen 
from  nitrates  and  nitrites. 

Fermentation. — Another  important  group  of  changes 
produced  by  micro-organisms  is  that  comprised  under 
the  comprehensive  title  of  "  fermentation,"  of  which  it  is 
difficult  to  give  an  accurate  definition,  for  the  distinction 
between  it  and  other  chemical  changes  due  to  the  activity 
of  micro-organisms  is  conventional  rather  than  scientific. 
The  original  conception  of  the  term  involved  the  occur- 
rence of  frothing  of  the  fermenting  liquid,  owing  to  the 
escape  of  gaseous  products.  Fermentation  is  brought 
about  by  the  action  of  ferments,  two  classes  of  which  are 
recognised,  viz.  the  living  or  organised  ferments,  which, 
in  other  words,  are  micro-organisms  ;  and  the  unorganised 
or  chemical  ferments,  bodies  such  as  pepsin,  which  in 
minute  amount  produce  changes  in  a  considerable  quantity 
of  the  substance  acted  upon,  without  themselves  under- 
going alteration. 

It  is  better  to  reserve  the  term  "  fermentation  "  for 
the  changes  brought  about  by  the  organised  ferments  or 
living  organisms,  and  to  call  the  unorganised  ferments 
enzymes,  and  the  changes  which  they  produce  zymolysis. 
As  fermentations  are  investigated  more  critically,  the 
tendency  is  to  find  that  they  are  brought  about  by  enzymes, 
extra -cellular  or  intra-cellular,  so  that  in  course  of  time 
this  distinction  may  no  longer  hold  good. 

1  Proc.  Linn.  Soc.  N.S.W.,  xxxvii,  1913,  p.  655. 


FERMENTATION  35 

The  following  are  the  chief  varieties  of  fermentation  : 
The  alcoholic  fermentation. — This  is  mainly  brought 
about  by  the  decomposition  of  sugars  of  the  hexose  group 
(C6H1206),  principally  dextrose  and  Isevulose,  by  yeasts 
into  alcohol  and  carbonic  acid,  but  some  of  the  bacteria 
and  moulds  also  produce  appreciable  quantities  of  alcohol. 
Other  carbohydrates  by  the  action  of  enzymes  secreted  by 
the  organisms  may  be  converted  into  hexoses,  which  are 
then  fermented.  The  general  reaction  is  as  follows  : 

C6H1206  =  2C2H60  +  2C02. 

As  a  matter  of  fact  small  amounts  of  by-products 
appear  in  addition  to  the  alcohol  and  carbonic  acid,  viz. 
glycerin,  succinic  acid,  and  higher  alcohols.  Until  1897 
no  enzyme  had  been  obtained  which  would  carry  out  this 
change  ;  it  only  occurred  when  the  living  yeast-cells  were 
present,  but  in  that  year  Buchner,  by  grinding  up  the 
living  yeast-cells,  obtained  a  juice  which  decomposed 
dextrose  with  the  formation  of  alcohol  and  carbonic  acid. 
This  "  zymase  "  Buchner  claimed  to  be  the  alcoholic 
enzyme  of  yeast. 

The  lactic  acid  fermentation. — This  is  brought  about 
chiefly  by  bacteria.  Hexoses  are  converted  into  lactic 
acid,  the  reaction  being 

C6H1206  =  2(HC3H603), 

kit  it  is  probably  not  actually  so  simple  as  this,  for 
carbonic  acid  is  given  off  at  the  same  time.  A  familiar 
example  of  this  form  of  fermentation  is  the  souring  of 
milk,  in  which  the  lactose  is  acted  upon  as  follows  : 

C12H22On  +  H20  =  4C3H603. 

The  butyric  acid  fermentation. — Butyric  acid  is  formed 
from  carbohydrates  by  the  action  of  bacteria,  mainly  the 
Bacillus  butyricus  and  Clostridium  butyricum,  the  latter  an 
anaerobic  organism,  some  by-products  being  formed  in 


36  A  MANUAL  OF  BACTERIOLOGY 

addition.  Milk  which  has  been  just  boiled  usually  under- 
goes the  butyric  rather  than  the  lactic  fermentation,  the 
spores  of  the  butyric  organisms  surviving.  Lactic  acid  is 
first  formed,  and  this  is  then  converted  into  butyric  acid  : 


2C3H603  -  C4H802  +  2C02  +  2H 


The  acetic  acid  fermentation.  —  The  conversion  of  alcohol 
into  acetic  acid  is  also  due  to  bacteria,  familiar  examples 
of  which  are  the  souring  of  beer  and  wine. 

Bacterial  enzymes.  —  Many  changes  brought  about  by 
bacteria  and  other  micro-organisms  are  due  to  enzymes, 
which  may  be  not  only  intra-cellular  but  may  escape  from 
the  cells  into  the  medium  in  which  they  are.  The  most 
familiar  example  is  the  peptonising  enzyme  produced  by 
bacteria  which  liquefy  gelatin  and  digest  coagulated 
protein,  fibrin,  etc.  The  enzymes  differ  :  an  organism 
which  liquefies  gelatin  does  not  necessarily  digest  blood- 
serum.  The  proteolytic  enzyme  is  tryptic  in  nature  and 
escapes  from  the  cells  into  the  surrounding  medium,  so 
that  some  of  the  liquefied  gelatin  free  from  cells  or  in 
which  their  action  is  inhibited  by  an  antiseptic,  liquefies 
other  gelatin  if  added  to  it.  Amylolytic  enzymes  are 
also  produced,  such  as  amylase  (digesting  starch),  maltase, 
lactase,  inulase,  and  invertase.  Lipases  and  rennet-like 
enzymes  also  occur.  "  Fermentation  "  of  urea  takes 
place  by  means  of  an  enzyme  secreted  by  the  Micrococcus 
ureoe,  etc.,  with  the  formation  of  ammonium  carbonate. 
These  enzymes  do  not  seem  to  possess  any  poisonous 
action. 

Formation  of  pigment.  —  Numerous  organisms,  especially 
those  of  air  and  water,  during  their  growth  produce  various 
coloured  pigments.  They  are  termed  "  chromogenic 
bacteria,"  examples  of  which  are  the  Sarcina  lutea  and 
Micrococcus  cereus,  var.  flavus,  which  form  citron-yellow 
pigments  ;  the  Bacillus  prodigiosus  and  Spirillum  rubrumt 


PIGMENT  FORMATION  37 

red  pigments  ;  the  Bacillus  violaceus  forms  a  rich  violet 
one  ;  and  the  Bacillus  pyocyaneus,  a  blue.  A  large  number 
of  chromogenic  organisms  require  oxygen  for  the  production 
of  the  pigment,  and  potato  is  often  the  most  favourable 
culture  medium.  In  some  cases  the  medium  may  become 
coloured,  and  the  property  of  fluorescence  be  conferred 
upon  it,  as  is  the  case  with  the  Bacillus  fluorescens  lique- 
faciens.  Usually  the  pigment  is  extra-cellular,  occasionally, 
as  in  B.  violaceus,  it  is  intra-cellular. 

A  group  of  organisms  producing  purplish  pigments  has 
been  described  under  the  name  of  "  purple  bacteria." 
It  is  doubtful  if  these  organisms  are  true  bacteria,  and  the 
pigment  may  exercise  a  respiratory  function  analogous 
to  chlorophyll. 

Phosphorescence,  or  light-production,  is  developed  by 
some  bacteria,  notably  by  many  marine  forms,  and  is 
well  seen  in  decomposing  fish.  Some  spirilla  are  also 
known  occasionally  to  produce  phosphorescence. 

A  necrotic  action  on  the  tissues  is  produced  by  many 
pathogenic  organisms.  For  example,  the  tubercle  and 
glanders  bacilli  cause  necrosis  and  caseation  of  the  sur- 
rounding tissues. 

Gas  production. — This  is  common  to  many  organisms. 
The  gas  may  consist  of  carbonic  acid,  hydrogen,  or  marsh 
gas,  and  in  some  cases  of  foul-smelling  sulphur  compounds, 
sulphuretted  hydrogen,  mercaptans,  etc. 

Sulphuretted  hydrogen  may  be  detected  by  the  blackening  of 
lead  acetate  paper.  Methyl  mercaptan  may  be  detected  by 
aspirating  a  current  of  air  through  the  culture,  through  a  calcium 
chloride  drying-tube,  and  then  through  a  test-tube  or  small  flask 
containing  isatin  dissolved  in  concentrated  sulphuric  acid.  The 
red  colour  of  the  isatin  solution  is  changed  to  olive-  or  grass-green 
by  the  mercaptan. 

Toxic  bacterial  products. — Almost  without  exception  the 
pathogenic  action  of  bacteria  is  brought  about  by  means 


38  A  MANUAL  OF  BACTERIOLOGY 

of  the  chemical  substances  produced  in  one  way  or  another 
by  their  metabolic  processes  (see  also  p.  24).  The  toxic 
bacterial  products  may  be  classified  as  follows  : 

(1)  Decomposition  products. — These  are  substances  pro- 
duced by  the  decomposition  of  the  medium  upon  which 
the  bacteria  are  growing.  Thus  proteoses  appear  to  be 
formed  by  the  anthrax  bacillus  and  the  pyogenic  cocci. 

The  ptomines  form  another  group  of  these  substances. 
These  are  a  very  important  group  of  nitrogenous  bodies, 
analogous  to  the  vegetable  alkaloids  and  mostly  solid  and 
crystalline  in  nature,  which  are  formed  by  the  action  of 
bacteria  on  protein  and  albuminoid  matter.  They  often 
occur  naturally  in  decomposing  and  putrefying  food, 
meat,  fish,  etc.,  and  as  many  of  them  are  virulent  poisons 
they  are  of  considerable  practical  import.  Poisoning  by 
tainted  food  may  be  due  to  the  absorption  of  such  toxic 
ptomines,  and  this  form  of  food-poisoning  is  known  as 
ptomine  poisoning.  A  number  of  toxic  ptomines  were 
isolated  by  Brieger  from  cultivations  of  pathogenic 
microbes,  and  great  importance  was  once  attached  to 
them.  They  are  referred  to  in  the  descriptions  of  the 
various  pathogenic  organisms. 

Brieger 's  work,  however,  needs  revision,  for  his  methods 
were  not  such  as  to  exclude  alteration  by  the  reagents 
employed. 

Stevenson  obtained  traces  of  a  highly  poisonous  crys- 
talline ptomine  from  some  sardines  that  had  caused  death. 
Vaughan  isolated  a  body,  tyrotoxicon,  apparently  identical 
with  diazobenzene,  from  poisonous  cheese  and  milk.  It 
seems  to  be  developed  by  the  action  of  organisms  belonging 
to  the  B.  coli  or  B.  lactis  aerogenes  types.  Mytilotoxin 
(C6H35N02)  is  the  specific  poison  of  toxic  mussels.  Such 
mussels  have  invariably  been  subjected  to  sewage  pollu- 
tion and  the  poison  is  probably  produced  by  the  action  of 
bacteria  derived  from  sewage.  Neurin  and  muscarin  are 


TOXINS  39 

extremely  poisonous  and  may  occur  in  decomposing 
flesh.  Some  of  the  ptomines  produced  by  putrefaction 
are  very  similar  to  certain  vegetable  alkaloids  and  are 
thus  of  considerable  medico-legal  importance.  The 
ptomines  are  not  specific  like  the  true  toxins,  and  toxic 
ones  may  be  produced  by  non-pathogenic  bacteria. 

(2)  Toxins. — These  are  the  soluble  poisons  elaborated 
by  the  bacteria  and  excreted  by  the  cells  into  the  sur- 
rounding medium.     They  are  regarded  by  Martin  and 
others  as  being  allied  to  the  proteoses.     Roux  and  Yersin 
suggested  that  the  diphtheria  poison  might  be  an  enzyme, 
while  Brieger  and  Frankel  regard  it  as  albuminous.     The 
toxins    are   non-basic  substances   closely  related  to   the 
proteins  and  hence  have  been  named  tox-albumins,  and 
are   considered  to   be  the   specific  toxic   poisons   of  the 
pathogenic  bacteria.     It  is  difficult  or  impossible  to  prepare 
them  in  a  state  of  purity  and  their  chemical  constitution 
is   therefore    unknown,   and  they   are   characterised   by 
extreme  specificity.     Such  are  the  poisons  of  the  diphtheria 
and  tetanus  bacilli. 

(3)  Endotoxins. — -These  are  toxic  substances  elaborated 
by  the  bacteria  which  do  not  to  any  extent  escape  from 
the  cells.     They  are  as  specific  as  the  toxins  and  possess 
analogous  properties  (see  below). 

(4)  Bacterial  proteins.- — These  are  toxic  constituents  of 
the  bacterial  cells  which  do  not  diffuse  from  the  cells,  are 
not  specific,  and  which  probably  usually  play  little  part 
in  the  production  of  the  disease  symptoms. 

LITERATURE 

On  Nitrification,  see  Warington,  Journ.  Chem.  Soc.,  1886,  et  seq.  ; 
Frankland,  Cantor  Lectures,  1892  ;  Nature,  1890,  et  seq.  ;  Lohnis, 
Handbuch  der  landwirtschaftlichen  Bakteriologie  (Borntraeger,  Berlin, 
1910,  full  bibliography).  On  Bacterial  Products,  see  Cellular  Toxins, 
by  Vaughan  and  Novy,  1902  (Bibliog.),  Ueber  Ptomaine,  by  Brieger, 
1885  ;  Macfadyen,  The  Cell  as  the  Unit  of  Life  (Churchill,  1908) ; 


40  A  MANUAL  OF  BACTERIOLOGY 

Wells,  C/nniinil  J'al/toluijy,  11)07;  Jlcwlell,  Ail.  "  Toxinfl  and 
Antiloxins,"  Thorpe's  Diet,  of  Chemistry,  1J)I.*{.  Kor  Cicncial 
Bibliography,  HOC  Kollo  and  Wassci maim,  ratkuycntn  J\l ikroor- 
yaniarncn. 

Endotoxins 

The  majority  of  pathogenic  micro -organisms  do  not  excrete  any 
appreciable  amount  of  toxin  ;  the  toxin  remains  within  the  cells. 
To  such  an  intra-cellular  toxin  the.  name  ol'  "  endotoxin  "  has  been 
given.  The  toxins  of  the  staphylococci  and  streptococci,  UK- 
typhoid-colon  group,  plague,  cholera,  etc.,  arc  cndotoxins.  Various 
methods  have  been  employed  to  prepare  these  endotoxins,  such  as 
extraction  of  the  cells  by  the  action  of  weak  alkalies  and  enzymes, 
and  by  autolysis  or  self-digestion. 

The  late  l)r.  Allan  Maefadyen  conceived  that  if  the  infra- 
oellular  toxins  (endotoxins)  of  such  organisms  us  the  typhoid 
bacillus,  cholera  vibrio,  etc.,  could  bo  obtained  free  1'rom  the 
bacterial  cells,  it  might  be  possible  to  prepare  sera  (anti-endoto.xic 
sera)  of  much  more  therapeutic  potency  than  the  ordinary  anti- 
microbic  sera. 

The  disintegration  of  the  bacterial  cells  in  the  presence  of  intense 
cold,  to  prevent  chemical  change  in  the  bacterial  juice  obtained, 
was  the  method  devised  by  J^lacfadyen  to  atla.in  (Ins  end.  With 
the  aid  of  his  colleagues,  Mr.  Rowland  and  Mr.  Barnard,  and  ol  his 
laboratory  assistants,  Messrs.  Burgess  and  Thompson,  apparatus 
and  methods  were  evolved  to  ollect  this. 

By  growing  on  the  surface  of  agar  or  other  suitable  medium  in 
plate  bottles  (Fig.  16),  scraping  oil  the  growth  and  suspending  this 
in  salt  solution,  ccntrifugalising  at  high  speed,  and  collecting  the 
bacterial  cell-mass  on  the  walls  of  the  centrifuge  vessels,  suilicient 
material  is  readily  obtained  to  grind  or  triturate,  and  thus  dis- 
integrate the  bacterial  cells  so  as  to  liberate  their  contents.  This 
is  accomplished  by  means  of  a  special  machine,  the  essential  part  ol 
which  consists  of  a  metal  cone  revolving  at  a  high  speed  in  a  metal 
pot,  the  bottom  of  which  is  shaped  so  as  to  lit  the  cone.  The  pot, 
with  its  contents,  is  immersed  in  a  vessel  of  liquid  air  or  other 
freezing  mixture,  and  the  bacterial  mass  is  ground. 

After  grinding,  the  ground  material  is  made  up  with  distilled 
water  or  with  0*1  per  cent,  sodium  hydrate,  so  as  to  form  a  10  per 
cent,  solution  (calculated  on  the  original  weight  of  the  moist 
bacterial  paste);  this  is  centrifugalised,  and  the  lluid  is  liltend 
through  a  sterile  Berkefeld  lilt rr. 


ENDOTOXINS 


42  A  MANUAL  OF  BACTERIOLOGY 

The  filtrate  thus  obtained  is  the  endotoxin,  and  is  used  to 
immunise  horses  and  other  animals  in  the  same  manner  as  with 
any  other  toxin  ;  it  should  be  used  as  fresh  as  possible.  The 
amounts  of  a  typhoid  or  cholera  endotoxin  employed  for  immunising 
must  at  first  be  small,  O2-0-5  c.c.,  as  it  produces  considerable 
disturbance  on  injection,  and  the  amount  is  gradually  increased. 
After  some  weeks'  treatment  a  dose  of  20-30  c.c.  may  be  injected. 
When  tests  show  that  the  serum  has  attained  the  necessary  potency, 
the  horse  is  bled  and  the  serum  obtained  and  bottled. 

The  endotoxins  also  possess  immunising  properties  to  a  high 
degree,  and  may  be  used  as  prophylactic  or  as  curative  vaccines ; 
they  markedly  raise  the  opsonic  index. 

Another  machine  has  been  devised  by  Barnard  for  disintegrating 
bacterial  and  other  cells.  It  is  supplied  by  Messrs.  Baker,  of  High 
Holborn,  and  is  depicted  in  Fig.  1,  p.  41. 

The  containing  vessel  consists  of  a  phosphor-bronze  body,  A,  in 
which  five  hardened  steel  balls,  B,  are  placed.  The  shape  of  the 
containing  vessel  is  such  that  when  these  balls  are  at  its  periphery 
they  accurately  fit  the  inner  side  of  the  vessel.  The  balls  are  evenly 
distributed  round  the  vessel  by  means  of  a  cage,  c,  and  during 
the  time  they  are  running  this  cage  ensures  that  they  are  equi- 
distant and  do  not  collide  one  with  another.  At  the  centre  of  the 
metal  vessel  is  a  steel  cone,  D,  which  is  of  such  a  size  that  it  keeps 
the  balls  in  their  proper  position  in  close  contact  with  the  periphery 
of  the  containing  vessel.  The  vessel  is  closed  by  a  screw  cap,  E, 
through  which  the  steel  cone  passes,  and  in  which  it  is  free  to 
rotate.  Over  the  whole  of  this  a  metal  cylinder,  r,  is  placed,  and 
is  screwed  down,  completely  sealing  the  upper  opening  in  the  metal 
vessel.  In  the  top  of  this  metal  cylinder  a  steel  bearing,  G,  is 
placed,  which  has  freedom  of  movement  in  a  horizontal  direction, 
but  is  kept  down  on  the  top  of  the  steel  cone  by  the  action  of  a 
spring.  It  therefore  follows  that  when  this  metal  cylinder  is 
screwed  down  the  steel  cone  is  pressed  on  to  the  balls,  and  the  balls 
are  in  their  turn  forced  out  to  the  periphery  of  the  metal  pot.  The 
whole  appliance  is  mounted  on  a  cone,  H,  and  a  centre,  I,  which 
are  carried  by  two  uprights  attached  to  the  base  plate,  J  ;  one  end 
of  the  shaft  is  attached  to  the  electric  motor. 

The  grinding  action  is  brought  about  by  retarding  the  revolu- 
tion of  the  central  cone,  D.  This  has  been  effected  by  mounting 
on  the  spindle  of  the  central  steel  cone,  D,  a  semi-cylindrical  mass  of 
iron  or  lead,  K,  the  weight  of  which  must  be  such  that  when  the 
whole  apparatus  is  rotated  it  is  sufficient  to  hold  the  central  cone 
still. 


ENDOTOXINS  43 

By  retarding  the  cone  in  this  way  a  drag  is  placed  on  the  balls, 
they  slide  to  a  certain  extent  over  the  inner  surface  of  the  pot  and 
exert  a  grinding  action. 

See  Hewlett's  Serum  Therapy,  1910  ;  Hewlett,  Proc.  Ray.  Soc., 
B,  1909  and  1911  ;  Proc.  Roy.  Soc.  Med.,  vol.  iii,  1909-10  (Patho- 
logical Section),  p.  165  ;  Barnard  and  Hewlett,  Proc.  Roy.  Soc.,  B., 
1911. 


CHAPTER  II 

METHODS  OF  CULTIVATING  AND  ISOLATING 
ORGANISMS 

IT  is  necessary  for  the  satisfactory  study  of  micro-organisms 
in  their  relation  to  the  various  processes  of  infection  and 
disease,  of  fermentation,  putrefaction,  and  the  like,  to 
separate  and  isolate  the  different  species  occurring  in  a 
mixture,  and,  having  done  so,  to  cultivate,  grow,  or 
propagate  each  species  on  suitable  soils  through  successive 
generations.  A  slight  consideration  will  show  that  unless 
we  work  with  pure  cultures — that  is,  cultures  consisting 
of  a  single  species — we  can  never  be  sure  that  a  particular 
result  is  due  to  a  given  organism ;  in  a  mixture  several  or 
all  of  the  forms  present  may  conduce  to  the  effect  pro- 
duced. With  regard  to  the  pathogenic  organisms,  or 
disease  germs,  Koch  laid  down  certain  conditions  which 
have  been  termed  "  Koch's  Postulates  "  (p.  147),  which 
must  be  complied  with  before  the  relation  of  an  organism 
to  a  disease  process  can  be  said  to  be  completely  demon- 
strated, one  of  which  is  that  "  the  organism  must  be 
isolated  and  cultivated  outside  the  animal  body  on  suitable 
media  for  successive  generations." 

In  order  to  isolate  organisms  in  a  state  of  purity  it  is 
absolutely  necessary  to  employ  vessels,  instruments,  and 
culture  media  which  are  sterile,  that  is,  free  from  any 
living  organisms,  and  to  possess  the  means  of  manipu- 
lating them  in  such  a  way  that  the  entrance  of  organisms 
from  without  is  prevented  and  contamination  avoided. 

44 


METHODS  OF  CULTIVATION 


45 


Various  methods  of  destroying  and  of  getting  rid  of 
organisms  are  known,  such  as  the  use  of  chemical  "  germi- 
cides," heat,  and  filtration  through  porous  porcelain. 
The  addition  of  chemical  germicides,  such  as  carbolic  acid 
or  corrosive  sublimate,  is  out  of  the  question  ;  for  although 
the  vessels  and  media  might  be  rendered  sterile  thereby, 


FIG.  2. — Hot-air  steriliser. 

the  growth  of  the  organisms  which  are  being  investigated 
would  equally  be  prevented,  so  that  the  two  last,  viz.  heat 
and  filtration,  are  those  which  are  employed,  the  former 
being  used  for  vessels,  instruments,  and  culture  media, 
solid  and  fluid,  the  latter  for  fluid  culture  media  only. 

Various  apparatus  are  needed  for  sterilisation  and  the 
preparation  of  culture  media.  These  will  now  be  described. 

Hot-air  steriliser  (Fig.  2).— This  is  a  rectangular  box  of 
sheet  iron  or  copper  with  double  walls,  having  an  air- 


46  A  MANUAL  OF  BACTERIOLOGY 

space  of  nearly  an  inch  between  them,  and  furnished 
with  a  door.  The  joints  should  be  brazed,  riveted,  or 
folded,  not  soldered.  The  outer  skin  at  the  bottom  should 
have  a  large  hole  cut  in  it  in  which  a  loose  piece  of  sheet 
iron  or  copper  should  be  inserted  to  protect  the  inner 
skin  from  oxidation  and  may  be  renewed  as  it  "  burns  " 

away.  The  top  is  perforated 
with  a  couple  of  holes,  through 
one  of  which  a  chemical  thermo- 
meter, registering  to  200°  C.,  is 
inserted  in  a  cork,  while  through 
the  other  some  form  of  mercurial 
regulator  can  be  introduced  if 
required,  but  is  not  usually 
needed.  In  the  hot-air  steriliser 
all  thin- glass  vessels  and  cotton- 
wool are  sterilised  by  heating  to 
a  temperature  of  about  150°  C. 
by  means  of  a  Bunsen  or  a  small 
ring  burner  under  the  steriliser, 
which  is  supported  on  a  suitable 
iron  stand.  If  the  steriliser  is 
placed  on  a  table  or  other 
wooden  support,  a  piece  of  sheet 

FIG.  3.— Steam  steriliser.  .  , 

iron,     asbestos     cardboard     or 

uralite  should  be  laid  over  the  wood  to  protect  it  from 
the  heat.  An  inexpensive  substitute  for  the  hot-air  steri- 
liser may  readily  be  devised,  any  iron  box  or  even  a 
biscuit- tin  being  used  for  the  purpose. 

Steam  steriliser  (Fig.  3). — This  consists  of  a  cylindrical 
or  rectangular  vessel  of  tinplate,  galvanised  iron,  or 
copper,  covered  on  the  outside  with  a  layer  of  felt  or 
asbestos,  having  a  false  perforated  bottom  supported  a 
few  inches  above  the  true  bottom,  and  provided  with  a 
movable  lid.  In  the  steam  steriliser  or  "  steamer  "  the 


AUTOCLAVE 


47 


culture  media,  and  thick  glass  vessels  and  other  apparatus 
which  would  crack  or  be  damaged  by  the  high  temperature 
of  the  hot-air  steriliser,  are  sterilised  by  steam.  The 
lower  chamber  of  the  steamer,  below  the  false  bottom, 
is  partly  filled  with  water,  which  is  boiled  by  means  of 
a  Bunsen  or  ring  burner.  Above  the  false  bottom  the 
culture  media  or  apparatus  are 
placed,  and  are  sterilised  by  the 
steam  at  100°  C.  which  fills  this 
space. 

Here  again  an  inexpensive  sub- 
stitute may  be  devised ;  the 
ordinary  kitchen  saucepan  with 
steamer  will  do  well  for  many 
purposes,  while  a  "  warren  pot  " 
answers  admirably. 

Autoclave  (Fig.  4). — This  is 
most  useful  for  many  purposes, 
but  it  is  expensive  and  not  a 
necessity,  as  the  steam  steriliser 
will  serve  almost  every  purpose 
for  which  the  autoclave  is  em- 
ployed with  the  expenditure  of 
a  little  more  time  and  trouble. 
It  consists  of  a  strong  boiler  of 
brass  or  gun-metal  with  a  removable  lid,  which  is  attached 
to  the  boiler  by  means  of  screw-bolts.  The  lid  is  provided 
with  a  safety  valve,  a  gauge  for  indicating  the  pressure 
and  temperature,  and  a  stopcock  to  relieve  the  pressure 
if  required.  A  small  quantity  of  water  is  placed  in  the 
bottom,  and  the  media  or  apparatus  to  be  sterilised  having 
been  introduced,  the  lid  is  screwed  down.  It  is  heated 
by  means  of  one  or  more  Bunsen  burners,  which  are 
turned  down  when  the  required  temperature  has  been 
reached.  The  temperature  usually  employed  is  about 


FIG.  4. — Autoclave. 


48  A  MANUAL  OF  BACTERIOLOGY 

115°  to  125°  C.  When  sterilising  media  care  should  be 
taken  that  the  vessels  are  not  too  full,  and  that  the  auto- 
clave is  allowed  to  cool  down  below  100°  C.  before  opening 
the  stopcock,  or  some  of  the  contents  may  be  lost  by 
violent  ebullition.  While  the  temperature  is  rising,  the 
stopcock  should  always  be  left  open  until  steam  is  being 
freely  generated  so  that  the  air  may  be  expelled. 

Air-pump. — An  exhaust  pump  is  useful  for  many  pur- 
poses, such  as  evaporating  to  dryness  in  vacuo,  filtration 
through  porous  porcelain  filters,  etc.  Any  form  will  serve, 
but  of  the  more  elaborate  ones  the  Fleuss  pump  (Fig.  5, 
p.  50)  made  by  the  Pulsometer  Engineering  Company  is 
one  of  the  best.  In  using  it  care  must  be  taken  that  no 
fluid  or  moisture  gains  access  to  the  barrel ;  to  avoid 
this  the  connecting  pipe  may  be  intercepted  with  a  vessel 
containing  strong  sulphuric  acid  (D,  Fig.  5),  over  the 
surface  of  which  the  exhausted  air  has  to  pass.  A  double- 
necked  Woulfe's  bottle  is  suitable  for  this,  the  inlet  and 
outlet  tubes  extending  nearly  down  to,  but  not  dipping 
below,  the  surface  of  the  sulphuric  acid. 

For  greasing  the  vessels,  etc.,  to  make  air-tight  joints, 
beeswax  dissolved  in  the  Fleuss  pump  oil  with  the  aid  of 
heat  to  a  stiff  paste  is  a  good  composition,  or  the  resin 
ointment  of  the  Pharmacopoeia  may  be  used.  Another 
good  grease  is  made  by  melting  together  one  part  of  black 
rubber,  one  part  of  vaseline,  and  one- third  part  of  paraffin 
wax. 

Centrifuge. — A  small  centrifuge  holding  two  or  four 
10  c.c.  tubes  is  a  necessity  in  the  laboratory.  A  form 
driven  by  hand  may  be  used,  but  one  driven  by  water  or 
electricity  is  almost  essential.  If  milk  is  examined,  a 
centrifuge  driven  by  power  and  containing  two  or  more 
tubes  having  a  capacity  of  not  less  than  70  c.c.  each  is 
required.  Many  forms  of  centrifuges  are  supplied  by 
Messrs,  Hearson, 


FILTERS  49 

Bell-jars  with  ground  rims  and  one  or  two  tubules  are 
useful  for  evaporation  in  vacuo.  They  should  stand  on  a 
square  of  thick  ground  glass.  To  make  an  air-tight  joint 
the  surface  of  the  rim  of  the  bell- jar,  which  must  be  quite 
clean,  should  be  well  greased  and  pressed  thoroughly 
home  on  the  ground-glass  plate.  A  thick  ridge  of  grease 
should  then  be  plastered  all  round  the  angle  formed  by  the 
rim  of  the  bell- jar  and  the  glass  plate.  Thick  rubber 
pressure  tubing  must  be  used  for  connections,  and  all 
joints  should  be  well  greased.  For  evaporating  large 
quantities  of  fluid  the  writer  devised  a  copper  stand  with 
shelves,  the  shelves  supporting  glass  dishes  containing 
alternately  strong  sulphuric  acid  and  the  fluid  to  be 
evaporated,  the  whole  being  placed  under  a  suitable 
bell- jar.  A  mercurial  gauge  is  a  useful  addition  to  show 
the  amount  of  exhaust  and  the  occurrence  of  leakage. 
The  ordinary  glass  filter  pumps  used  in  chemical  work 
and  actuated  by  a  stream  of  water  are  also  useful  for  many 
purposes. 

Porous  porcelain  filters. — The  forms  which  are  generally 
employed  are  the  Pasteur- Chamberland,  the  Doulton,  and 
the  Berkefeld.  These  consist  of  "  candles  "  composed  in 
the  first  two  of  unglazed  porous  porcelain,  in  the  last  of  a 
specially  prepared  diatomaceous  earth.  The  filtration 
through  the  Pasteur- Chamberland  is  much  slower  than 
through  the  Berkefeld.  All  give  a  germ-free  filtrate,  but 
the  last  should  be  employed  if  the  fluid  is  thick  or  contains 
many  particles ;  a  preliminary  filtration  through  paper 
is  an  advantage.  A  useful  method  of  conducting  filtration 
is  the  following  :  The  filter  "  candle  "  (B,  Fig.  5,  p.  50) 
is  connected  by  a  short  length  of  pressure  tubing  with  a 
piece  of  glass  tubing  passing  through  a  rubber  cork  in  the 
neck  of  an  ordinary  filtering  flask  c.  The  "  candle  "  is 
placed  in  a  jar  A,  such  as  a  glass  measure  or  urine- jar, 
which  is  filled  up  with  the  solution  to  be  filtered.  The 

4 


50 


A  MANUAL  OF  BACTERIOLOGY 


lateral  branch  of  the  filter  flask  is  then  connected  with  the 
air-pump.  On  exhausting,  the  fluid  passes  through  the 
filter  "candle"  over  into  the  filtering  flask;  in  which  it 
is  collected.  Before  use  the  "  candle "  should  be  well 
scrubbed  and  some  water  or  J  per  cent,  carbolic  run 
through  to  clean  it,  and  the  whole  may  be  sterilised  in  the 


FIG.  5. — Fleuss  exhaust  pump,  arranged  for  filtration. 

steamer  for  an  hour  or  two.  After  use  the  same  process 
should  be  repeated  to  cleanse  it. 

Flasks,  beakers,  and  test-tubes. — A  good  supply  of  these 
of  various  sizes  is  required  :  Erlenmeyer  and  ordinary 
shapes,  tall  and  short  forms  of  beakers,  etc.  A  few  "  yeast 
flasks  "  are  also  useful  (see  Fig.  13,  p.  75).  Beakers  and 
flasks  of  "  Jena  "  glass  are  to  be  preferred.  Enamelled 
iron  ware,  jugs,  saucepans,  mugs,  etc.,  may  replace  glass 
for  many  purposes. 

The  most  useful  size  of  test-tube  is  5"  x  f "  ;  a  fow 
larger  sizes  and  "  boiling  tubes  "  should  also  be  kept. 

Platinum  needles  (Fig.  6). — Two  or  three  platinum 
needles  are  required.  They  consist  of  about  two  inches 


PIPETTES  51 

of  platinum  wire  in  a  handle  of  glass  rod.  One  end  of 
a  glass  rod  is  softened  in  the  Bunsen  or  blowpipe  flame, 
and  about  an  eighth  of  an  inch  of  the  platinum  wire  is 
embedded  in  it  with  a  forceps,  the  wire  having  been  first 
heated  to  a  red  heat.  The  glass- wire  joint  is  then  well 
annealed  in  the  flame  and  allowed  to  cool  slowly.  Metal 
handles  may  also  be  used.  Two  thicknesses  of  platinum 
wire  are  desirable,  viz.  04  mm.  (27-28  B.W.G.)  for  most 
purposes,  but  a  thicker  wire  of  about  0-7  mm.  where 


FIG.  6. — Platinum  needles. 

stiffness  is  required,  and  one  or  two  3  in.  or  more  in  length 
are  useful. 

Forceps,  needles,  etc.— Several  forceps  are  necessary,  the 
ordinary  dissecting  form  in  two  or  three  sizes,  one  or  two 
pairs  of  fine  pointed,  two  or  three  small  brass  ones,  and 
two  or  three  pairs  of  the  "  Cornet "  pattern.  A  few 
ordinary  sewing  needles  of  various  sizes  mounted  in 
wooden  handles  serve  all  purposes. 

Glass  pipettes  and  capillary  tubes. — These  are  useful  for 
preserving  or  storing  blood  or  pus,  etc.,  for  examination, 
for  sterile  water  in  making  film  specimens,  and  for  many 
other  purposes.  A  blowpipe  worked  by  a  foot  bellows 
is  required  for  making  pipettes,  etc.  A  piece  of  glass 
tubing  is  heated  in  the  blowpipe  flame  until  quite  soft ; 
it  is  then  taken  out  of  the  flame  and  the  two  ends  are  pulled 
steadily  apart ;  this  forms  a  capillary  tube  of  greater  or 
lesser  length  and  smaller  or  larger  diameter,  and  it  can  be 
sealed  off  in  convenient  lengths.  To  make  a  pipette 
proceed  in  the  same  way  :  seal  off  the  capillary  tube  two 
or  three  inches  from  the  wide  tube,  then  heat  this  close 


52  A  MANUAL  OF  BACTERIOLOGY 

up  to  where  it  was  heated  before,  and  draw  out  again 
and  seal  off  two  or  three  inches  from  the  bulb.  In  this 
way  a  capillary  tube  with  a  bulb  at  its  middle  is  formed 
(Fig.  7).  "  Vaccine  tubes,"  pipettes  made  of  glass  tubing 
drawn  out  at  one  end,  and  Wright's  capsules  (see  Fig.  35, 
a  and  d,  p.  215)  are  also  useful. 

India-rubber  caps. — A  few  indiarubber  caps  for  capping 
test-tube   or   flask   cultures   are   required.     They   retard 


FIG.  7. — Glass  pipette. 

evaporation  and  the  desiccation  lof  the  medium,  and 
prevent  the  entrance  of  moulds.  For  use  they  should  be 
soaked  in  1-500  corrosive  sublimate  solution;  they 
should  not  be  kept  pn  the  solution,  as  vulcanised  rubber 
absorbs  mercuric  chloride  (Glenny  and  Walpole).  Tinfoil, 
gutta-percha  tissue  (sealed  down  by  warming),  paraffin 
wax,  sealing  wax,  or  plasticine  may  also  be  used  to  cover 
the  tops  of  tubes  and  flasks. 

Preparation  of  Sterile  Test-tubes,  Flasks,  etc., 
for  the  Reception  or  Manipulation  of  Cul- 
ture Media 

To  sterilise  cotton-wool. — Non-absorbent  cotton-wool, 
best  or  No.  2  quality,  is  used  for  plugging  purposes.  The 
wool  should  be  pulled  apart  so  as  to  assist  the  penetration 
of  heat ;  in  the  compressed  condition  the  interior  is 
difficult  to  sterilise  The  separated  wool  is  placed  in  the 
hot-air  steriliser  and  the  temperature  is  slowly  raised  to 
145°  C.  and  maintained  at  this  for  at  least  an  hour.  Above 
150°  C.  cotton-wool  becomes  brown  and  brittle.  It  is  a 
common  practice  now  to  use  various  coloured  wools  for 
the  different  culture  media,  especially  the  carbohydrate 


GLASSWARE  53 

ones,  so  that  they  are  readily  distinguishable  by  the  eye. 
The  coloured  wools  may  be  purchased,  or  the  ordinary 
white  wool  may  be  dyed  with  the  "  Dolly  "  dyes. 

Glass  vessels. — The  vessels  (usually  test-tubes,  flasks, 
and  dishes)  are  thoroughly  washed  and  rinsed  in  water, 
then  rinsed  with  25  per  cent,  hydrochloric  acid,  and 
afterwards  washed  well  with  tap-water  and  drained.  A 
final  rinse  with  distilled  water  or  alcohol  is  an  advantage, 
as  no  deposit  then  occurs  on  drying.  The  cleansed  vessels 
should  be  dried  before  sterilising,  either  in  the  air  or  by 
placing  in  the  hot-air  steriliser  for  half  an  hour.  When 
dry,  the  vessels  are  plugged  with  a  firm  plug  of  the  sterilised 
cotton-wool,  and  are  placed  in  the  hot-air  steriliser,  the 
temperature  of  which  is  then  raised  to  about  150°  C. 
They  should  remain  at  this  temperature  for  not  less  than 
half  an  hour,  after  which  the  steriliser  and  its  contents 
are  allowed  to  cool  slowly. 

Petri  dishes  for  plate  cultures,  graduated  pipettes,  etc., 
are  cleaned  as  described  for  tubes  and  flasks.  They  may 
be  sterilised  and  kept  in  sheet-iron  or  copper  boxes  of 
appropriate  size  and  shape. 

If  tubes,  flasks,  pipettes,  etc.,  are  required  in  a  hurry 
they  may  be  rapidly  sterilised  as  follows  :  After  washing 
in  water  they  are  rmsed  with  5  per  cent,  carbolic,  then 
with  absolute  alcohol,  and  finally  with  ether,  and  are  then 
well  flamed  over  a  Bunsen  flame,  holding  in  a  suitable 
forceps  or  holder.  The  ether  evaporates  and  burns  at 
the  mouth,  and  when  dry,  a  pledget  of  cotton-wool  is  held 
in  the  forceps  and  singed  in  the  flame,  and,  while  burning, 
the  tube  or  flask  is  plugged  with  it. 

When  thick  glass  vessels,  such  as  measures,  etc./  have 
to  be  sterilised,  it  is  not  safe  to  do  this  in  the  hot-air 
steriliser  unless  the  heating  and  cooling  are  carried  out 
very  slowly,  as  they  are  very  liable  to  crack.  It  is  prefer- 
able, after  cleaning  and  plugging  with  sterile  wool,  to 


54  A  MANUAL  OF  BACTERIOLOGY 

steam  in  the  steam  steriliser  or  the  autoclave,  the  heating 
and  cooling  being  conducted  slowly. 


Culture  Media 

The  ordinary  methods  of  preparing  culture  media  are 
here  given,  but  "  Standard "  media,  having  definite 
reactions,  are  now  largely  employed  (for  the  method  of 
standardisation,  see  p.  64).  Certain  special  media  will  be 
described  as  required.  In  all  cases  the  media  are  filled 
into  the  cleansed  and  sterilised  vessels,  test-tubes,  flasks, 
etc.  (p.  53).  For  ordinary  laboratory  cultures  test-tubes 
are  generally  used.  Media  which  are  solid  at  ordinary 
temperatures,  e.g.  agar,  gelatin,  and  serum,  are  prepared 
either  as  deep,  upright  tubes  (Fig.  8,  A),  for  which  8-15  c.c. 
of  the  medium  are  required  for  a  tube,  or  as  sloping  tubes 
(Fig.  8,  c),  for  which  4-5  c.c.  are  required  for  a  tube.  Of 
fluid  media  7-15  c.c.  are  used  for  a  tube.  The  prepared 
media  having  been  introduced  into  the  test-tubes,  etc., 
sterilisation  is  effected  in  the  steam  steriliser  (p.  46)  by 
steaming  for  twenty  to  thirty  minutes  on  two  or  three 
successive  days,  or  in  the  autoclave  (p.  47)  by  heating  to 
115°-120°  C.  for  half  an  hour  on  one  occasion.  Culture 
media  may  also  be  kept  in  bulk  in  flasks  ;  these  need 
somewhat  longer  sterilisation  than  tubes.  Tubes  of  some 
of  the  culture  media  can  also  be  purchased  ready  for  use. 
Certain  media  can  be  obtained  in  powder  form  (Chopping's) 
from  Messrs.  Baird  and  Tatlock,  and  in  tabloid  form 
(Thompson's)  from  Messrs.  Burroughs  and  Wellcome.  These 
are  convenient  when  small  quantities  are  required  for 
occasional  use. 

Acid  beef-broth. — The  basis  of  the  most  important 
culture  media,  viz.  peptone  beef-broth,  gelatin,  and  agar- 
agar,  is  an  infusion  of  meat  prepared  usually  from  beef. 
In  order  to  prepare  this  infusion,  which  may  be  termed 


CULTURE  MEDIA 


55 


acid  beef-broth,  proceed  as  follows  :  Take  1  Ib.  of  beef 
("  gravy  beef  ")  free  from  fat,  chop  fine  or  mince,  add  one 
litre  of  tap-water,  and  allow  it  to  simmer  in  a  saucepan 
for  one  hour  ;  cool,  remove  any  solidified  fat  from  the 
surface,  and  filter  through  filter-paper  into  a  clean  glass 
flask.  If  not  required  for  immediate  use,  plug  the  neck 
of  the  flask  with  cotton-wool,  and 
steam  in  the  steam  steriliser  (or  boil) 
for  three-quarters  of  an  hour  on  two 
successive  days.  It  may  then  be  kept 
until  required. 

Peptone  beef -broth. — Take  one  litre 
of  the  acid  beef-broth,  add  to  this 
10  grm.  of  peptone  (Witte's)  and  5 
grm.  of  common  salt  (i.e.  1  per  cent, 
peptone  and  0-5  per  cent,  sodium 
chloride),  mix  in  a  flask,  and  steam 
in  the  steam  steriliser  until  dissolved. 
When  dissolved,  remove  from  the 
steam  steriliser  and  render  slightly 
alkaline  with  a  10  per  cent,  solution 

of    caustic   soda    (preferably)   or    of 

,.  -,  77V,  FIG.  8. — Tubes  of  culture 

sodium  carbonate ,  glazed  litmus-paper      media    A  Upright  agar 
being  used  as  an  indicator.     Having      B.  Potato,    c.  Sloping 
done  this,  return  to  the  steamer  for      agar> 
one  hour,  then  filter  through  two  thicknesses  of  German 
filter-paper.     It  should  now  be  quite  clear  and  bright  and 
may  be  kept  in  bulk,  after  sterilising,  or  be  introduced 
into  test-tubes,  etc.,  and  sterilised.     Beef-broth,  if  pre- 
pared in  this  manner,  may  need  no  clarifying,  but  if  it 
should  filter  at  all  cloudy,  cool  to  50°  C.,  add  the  white  of 
an  egg  beaten  up  with  the  shell,  and  steam  for  half  an 
hour,   filter,  and  finally  sterilise   as   before.     Other  pre- 
parations of  peptone,  e.g.  Peptone  Chapoteaut,  may  be 
used. 


56  A  MANUAL  OF  BACTERIOLOGY 

Instead  of  infusion  made  from  meat,  meat  extracts  are  now 
commonly  used.  The  following  is  the  composition  of  "  Lemco  " 
broth : 

Lemco      .......     10-20  grm. 

Peptone  (Witte) 10-20  grm. 

Sodium  chloride         .....       5-10  grm. 

Water  (preferably  distilled)          ...  1  litre 

The  constituents  are  dissolved  with  the  aid  of  heat,  neutralised, 
clarified  and  filtered.  Lemco  may  also  be  used  to  make  all  the 
other  media  for  which  acid  beef-broth  is  employed. 

Veal-broth. — For  some  purposes  veal  presents  advan- 
tages over  beef,  e.g.  for  growing  the  tubercle  bacillus. 
When  obtained  from  the  butcher's  the  veal  is  frequently 
powdered  with  flour  ;  this  should  be  brushed  and  washed 
off  as  completely  as  possible,  as  it  renders  the  broth 
turbid  and  difficult  to  clarify.  The  veal-broth  is  made 
in  precisely  the  same  way  as  peptone  beef-broth.  It  is, 
however,  often  slightly  alkaline,  so  that  less  alkali  is 
required  for  neutralisation.  For  the  cultivation  of  the 
tubercle  bacillus  about  4  to  6  per  cent,  of  glycerin  should 
be  added. 

Glycerin  beef-broth  is  prepared  in  the  same  manner, 
4  to  6  per  cent,  of  the  best  glycerin  being  added  to  the 
fluid  after  filtration. 

Glucose  broth. — For  the  cultivation  of  anaerobic  organisms 
the  addition  of  0-5  to  2  per  cent,  of  grape  sugar  is  an 
advantage.  It  should  be  added  after  filtration. 

Egg  broth.— Besredka  and  Jupille1  describe  the  com- 
position of  this  as  follows  : 

White  of  egg  (10  per  cent,  solution)    .          .          .4  parts 
Yolk  of  egg  (10  per  cent,  solution)      .          .          .1  part 
Ordinary  nutrient  broth    .          .          .         .         .5  parts 

The  egg-white  is  beaten  up  with  ten  times  its  volume 
of  distilled  water,  filtered  through  cotton- wool,  heated 

1  Ann.  de  VInst.  Pasteur,  xxvii,  1913,  p.  1009. 


CULTUEE  MEDIA  57 

to  100°  C.,  and  filtered  through  "  papier  Chardin."  The 
liquid  is  tubed  and  sterilised  at  115°  C.  for  twenty  minutes. 
The  yolk  is  beaten  up  with  ten  times  its  volume  of  distilled 
water  and  a  sufficiency  of  normal  caustic  soda  solution 
is  added  to  clarify  it  (about  1  c.c.  per  100  c.c.).  It  is  then 
treated  as  the  egg-white.  The  authors  recommend  the 
use  of  Martin's  broth. 

Peptone  water. — Add  to  distilled  or  tap  water  1  to  2  per 
cent,  of  Witte's  peptone  and  \  per  cent,  of  common  salt, 
dissolve  by  heat,  make  faintly  alkaline,  steam  for  one 
hour  and  filter. 

For  the  cholera  vibrio  it  is  an  advantage  to  add  1  per 
cent,  instead  of  J  per  cent,  of  common  salt  (Dunham's 
solution). 

Beer-wort. — Procure  beer-wort  (preferably  unhopped) 
from  the  brewery.  Allow  it  to  stand  in  a  cool  place 
for  twelve  hours,  filter,  and  then  steam  for  an  hour  and 
filter  again.  Fill  into  sterile  test-tubes  and  sterilise. 

Nutrient  gelatin. — This  is  prepared  in  precisely  the  same 
manner  as  peptone  beef-broth  with  the  addition  of 
100  grm.  of  the  best  "  gold  label "  gelatin  (Coignet's)  per 
litre.  After  the  addition  of  the  egg,  steam  for  an  hour 
and  then  filter  through  two  thicknesses  of  filter-paper  in 
a  hot- water  funnel  (this  is  best,  but  it  may  be  done  in  the 
steamer  at  a  low  temperature,  e.g.  35°  C.).  Fill  into 
test-tubes  and  sterilise.  After  the  third  steaming  the  tubes 
are  allowed  to  solidify,  either  in  the  upright  or  oblique 
position,  according  as  they  are  required  for  stab  or  surface 
cultivation. 

In  hot  summer  weather  15  or  even  20  per  cent,  of  gelatin  (150  grm. 
or  200  grm.  to  the  litre)  are  necessary  for  the  product  to  remain 
solid,  as  nutrient  gelatin  melts  at  24°  C.  or  a  little  under.  Pro- 
longed boiling  diminishes  and  ultimately  destroys  the  gelatinising 
power  of  gelatin,  so  the  less  it  is  heated  the  better.  It  must  not 
be  autoclaved. 


58  A  MANUAL  OF  BACTERIOLOGY 

Glucose  gelatin. — Ordinary  gelatin  with  the  addition  of 
1  to  2  per  cent,  of  grape  sugar. 

Beer-wort  gelatin. — This  is  one  of  the  best  culture  media 
for  yeasts  and  some  of  the  fungi  (e.g.  ringworm).  Procure 
from  the  brewery  some  beer-wort,  preferably  unhopped, 
and  add  to  every  litre  100  grm.  of  gelatin.  Dissolve, 
clarify,  and  filter,  as  in  the  case  of  ordinary  gelatin.  It 
is  not  neutralised. 

Nutrient  agar-agar. — This  is  one  of  our  most  valuable 
culture  media,  and  has  the  advantage  over  nutrient 
gelatin  that  it  remains  solid  at  blood-heat. 

Agar  is  a  carbohydrate  substance  of  high  melting-point 
and  considerable  gelatinising  power,  obtained  from 
Eastern  seaweeds.  The  powdered  form  is  now  generally 
used.  Add  15  grm.  (i.e.  1J  per  cent.)  of  powdered  agar 
to  1  litre  of  acid  beef-broth,  together  with  10  grm.  of 
peptone  and  5  grm.  of  common  salt  in  a  large  glass  flask, 
place  in  the  water-bath  until  dissolved  (half  an  hour  to 
one  hour),  and  then  render  alkaline  as  for  peptone  beef- 
broth ;  allow  it  to  cool  to  50°  0.,  and  add  the  white  of 
an  egg.  Return  to  the  steamer  for  an  hour  and  a  half, 
then  filter  through  an  agar  filter-paper  ("  papier  Chardin  ") 
in  a  hot- water  funnel  or  in  the  steamer.  By  this  treatment 
a  litre  of  agar  should  pass  through  the  filter  in  two  to  three 
hours.  If  it  does  not  come  through  clear,  add  another 
white  of  egg  and  repeat  the  process. 

If  an  autoclave  is  available,  a  quicker  and  better  method 
is,  after  neutralising  and  adding  the  white  of  an  egg,  to 
place  in  the  autoclave  with  a  small  beaker  inverted  over 
the  mouth  of  the  flask,  and  heat  to  134°  C.  (two  atmospheres 
pressure)  for  half  an  hour.  Turn  the  gas  out,  and  allow 
to  cool  without  opening  the  stopcock.  When  cool,  open, 
and  filter  through  the  special  agar  filter-paper  in  a  hot- 
water  funnel ;  the  agar  will  pass  through  in  about  ten 
minutes  or  a  quarter  of  an  hour.  Fill  into  test-tubes  and 


CULTURE  MEDIA  59 

sterilise.  Solidify  in  the  upright  or  oblique  position  as 
required. 

In  the  case  of  bar  or  stick  agar,  first  steep  the  agar  in 
1  per  cent,  acetic  acid  for  a  quarter  of  an  hour,  then  drain 
and  wash  it  so  as  to  thoroughly  remove  the  acid.  The 
further  procedure  is  the  same  as  detailed  above.  This 
yields  a  very  clear,  pale  product,  and  is  perhaps  preferable 
when  an  autoclave  is  not  available. 

Glycerin  agar. — Add  4  to  6  per  cent,  of  glycerin  to 
the  nutrient  agar  after  filtration  and  proceed  as  before. 

Glucose  agar. — One  or  two  per  cent,  of  grape  sugar  is 
added  to  the  nutrient  agar  after  filtration. 

Litmus  media. — The  addition  of  neutral  litmus  to  the 
various  culture  media  is  a  useful  method  of  demons- 
trating the  production  of  acid  or  of  alkali  by  organisms. 
To  prepare  the  litmus  solution  take  the  lump  litmus, 
powder  finely,  and  boil  with  distilled  water  so  that  a 
saturated  solution  is  obtained.  Filter,  and  preserve  in 
a  flask  stoppered  with  cotton-wool,  after  sterilising  by 
boiling  for  half  an  hour  on  two  successive  days.  For 
some  purposes  a  special  solution  of  litmus,  the  Kubel- 
Tiemann  solution,  which  can  be  procured  ready  for  use,  is 
employed.  It  must  not  have  any  antiseptic  added  to 
it  (as  is  sometimes  done  to  preserve  it  for  use  in  the 
chemical  laboratory). 

Sufficient  of  this  litmus  infusion  is  added  to  the  nutrient 
media,  after  filtration,  to  tinge  them  a  distinct  purplish 
colour.  After  steaming  the  colour  has  usually  disappeared, 
but  returns  as  the  tubes  cool. 

Milk. — Use  separated  milk,  but  failing  this,  centri- 
fugalise  ordinary  new  milk,  or  place  it  in  a  tall  cylinder 
and  allow  it  to  stand  overnight  in  a  cool  place,  preferably 
in  an  ice  safe.  Then  pipette  off  the  milk  from  the  bottom, 
rejecting  the  cream.  Introduce  the  separated  milk  into 
test-tubes  to  the  depth  of  about  an  inch  to  an  inch  and 


60 


A  MANUAL  OF  BACTEEIOLOGY 


a  half  and  steam  for  one  hour  on  two  successive  days. 

The  milk  is  usually  tinged  with  litmus  before  tubing, 

forming  litmus  milk. 

Potatoes. — Choose  sound  potatoes,  and  scrub  them  well 

with  water  to  remove  dirt.  Cut  off  the  ends,  and  with  a 
C^*.  cork-borer,  slightly  smaller  than  the  test- 
tubes  which  are  used,  bore  through  the 
potato  so  that  a  cylindrical  piece  is  re- 
moved. Push  this  out  of  the  borer,  and 
divide  it  into  two  portions  by  a  very 
oblique  transverse  cut,  so  that  two  wedge- 
shaped  pieces  are  obtained,  and  in  this 
manner  prepare  as  many  pieces  as  there  are 
tubes  to  be  filled.  Place  them  in  a  basin 
under  the  tap,  and  allow  the  water  to  flow 
over  them  for  about  two  hours.  This  pre- 
vents the  darkening  of  the  potato  in  the 
subsequent  steaming,  as  does  also  the  use 
of  a  silver  borer.  The  test-tubes  for  the 
potato- wedges  are  prepared  as  follows : 
After  plugging  and  sterilising  in  the  ordi- 
nary way /introduce'  a  small  pledget  of  steri- 
lised wool  into  each,  push  to  the  bottom, 
and  moisten  with  a  little  sterilised  distilled 

^^         water.     Drop  the  potato-wedges  into   the 
FIG.  9. — Roux's  .    ,  i  T  .,.       ,  , 

tube  for  potato.  tubes>  Plug>  and  sterilise  by  steaming  for 
three-quarters  of  an  hour  on  two  succes- 
sive days  (Fig.  8,  B).  The  object  of  the  moist  wool 
is  to  prevent  drying,  and  for  the  same  purpose  Roux's 
tubes  (Fig.  9)  may  be  used,  the  lower  bulb  being  filled  with 
water. 

Blood-serum. — Clean  some  glass  jars  of  about  1  to 
3  litres  capacity,  plug  with  wool,  and  sterilise  in  the 
steamer  for  an  hour  on  three  successive  days.  Bleed 
a  horse,  with  aseptic  precautions,  and  catch  the  blood 


CULTURE  MEDIA  61 

in  these  sterilised  jars.  Allow  the  jars  to  stand  in  a 
cool  place  for  twelve  hours.  Then  pipette  off  the  clear 
serum  with  a  sterile  pipette,  and  fill  the  sterilised  test- 
tubes  to  the  depth  of  2-4  cm.  The  tubes  are  then 
arranged  in  a  sloping  position  on  the  shelves  of  the  serum 
inspissator,  or  failing  this  in  a  hot- water  oven,  the  tem- 
perature of  which  should  be  about  50°  C.  At  this  tempera- 
ture they  remain  for  thirty  hours ;  it  is  then  raised  to 
65°  C.,  at  which  temperature  the  serum  coagulates  in  from 
four  to  six  hours  and  the  tubes  are  now  ready  for  use.  It 
is  well,  however,  to  place  them  in  the  blood-heat  incu- 
bator for  a  night,  so  that  any  contaminating  bacteria  may 
form  colonies,  and  the  contaminated  tubes  may  then  be 
rejected. 

Lqffler's  blood-serum  is  prepared  by  adding  one  part 
of  glucose  broth  to  three  parts  of  the  serum  before  inspissa- 
tion. 

The  serum  inspissator  is  practically  an  incubator  (see 
p.  68)  with  slightly  inclined  (10-15°)  shelves,  on  which  the 
tubes  rest,  and  thus  the  serum  is  coagulated  in  a  sloping 
position. 

Fluid  serum,  etc. — Fluid  blood-serum,  ascitic  and 
hydrocele  fluids,  etc.,  are  sometimes  useful,  and  may  be 
used  alone  or  mixed  with  peptone  beef -broth  in  various 
proportions. 

Ascitic  or  hydrocele  fluid  may  be  obtained  by  using 
sterile  trocars,  etc.,  and  carrying  out  the  tapping  with 
aseptic  precautions,  collecting  the  fluid  in  sterilised  flasks. 
It  is  better  to  collect  in  several  small  flasks  than  in  one 
large  one. 

Fluid  blood- serum  may  be  obtained  by  collecting  blood 
with  aseptic  precautions  in  sterilised  flasks.  When  the 
blood  has  coagulated  and  the  serum  separated,  the  serum 
is  pipetted  off  with  a  sterile  pipette  into  sterile  flasks. 

The  flasks  of  serum,  etc.,  should  be  kept  in  a  warm 


62  A  MANUAL  OF  BACTERIOLOGY 

place  for  two  or  three  days  to  make  sure  that  they  are 
sterile,  those  in  which  a  growth  appears  being  rejected. 

Serum,  ascitic  fluid,  etc.,  may  also  be  obtained  sterile 
by  filtering  through  a  sterilised  Berkefeld  filter  into  sterile 
flasks. 

Serum,  ascitic  and  hydrocele  fluids,  etc.,  may  be  pre- 
served in  bulk  and  used  as  required.  The  material  is 
collected  as  aseptically  as  possible,  5  per  cent,  of  chloro- 
form is  added,  and  the  whole  is  well  mixed  and  kept  in  a 
cool  place  in  the  dark  in  a  well-stoppered  bottle.  Sub- 
sequently, during  the  process  of  sterilisation,  the  chloroform 
is  volatilised. 

Serum  agar  (Kanthack  and  Stevens). — Ascitic,  pleuritic, 
or  hydrocele  fluid  is  collected  in  clean  (not  necessarily 
sterilised)  flasks,  and  allowed  to  stand  overnight  in  a  cool 
place  to  allow  the  sediment  or  blood  to  deposit.  The 
clear  fluid  is  then  poured  off,  and  to  each  litre  enough 
of  a  10  per  cent,  caustic  potash  solution  is  added  to  render 
it  very  distinctly  alkaline — usually  about  2  c.c.  to  every 
100  c.c.  of  the  fluid.  The  alkaline  fluid  is  heated  in  the 
autoclave  for  two  to  four  hours.  To  this  fluid  1-5  to  2  per 
cent  of  agar  is  added,  and  the  mixture  is  heated  until  the 
agar  dissolves.  It  is  then  filtered,  introduced  into  test- 
tubes,  sterilised,  and  solidified  in  the  ordinary  way.  The 
addition  of  5  per  cent,  of  glycerin  and  1  per  cent,  of  glucose 
is  an  advantage. 

Serum  agar  may  also  be  prepared  by  adding  sterile 
serum  or  hydrocele  or  ascitic  fluid,  warmed  to  45°  C., 
to  sterile  nutrient  agar  (2  to  3  per  cent,  agar)  melted  and 
cooled  to  45°  C.  Equal  parts  of  the  serum  and  agar  may 
be  mixed,  or  1  part  of  serum  to  2  parts  of  agar. 

Blood  agar. — This  may  be  prepared  by  smearing  the 
surface  of  the  agar  in  sloping  agar-tubes  with  blood 
obtained  aseptically  from  the  finger  or  from  a  rabbit. 
Or  blood  obtained  aseptically  may  be  defibrinated  by 


CULTUKE  MEDIA  63 

shaking  with  glass  beads  or  with  a  coil  of  fine  wire,  and 
the  defibrinated  blood,  warmed  to  45°  C.?  is  added  to 
sterile  agar  liquefied  by  boiling  and  cooled  to  45°  C. 
Haemoglobin  agar  may  be  prepared  by  laking  defibrinated 
blood  by  the  addition  of  sterile  distilled  water  and  adding 
to  the  liquid  agar  as  before.  Blood  agar  cannot  be 
sterilised  after  preparation,  and  the  blood  therefore  must 
be  sterile. 

Alkali  albumen  (Lorrain-Smith). — To  100  c.c.  of  fresh 
serum  add  1  to  1*5  c.c.  of  a  10  per  cent,  caustic  soda 
solution  ;  mix  and  introduce  into  test-tubes  in  the  ordinary 
way.  Place  the  test-tubes  in  the  slanting  position  in  the 
autoclave  at  115°  C.  for  twenty  minutes,  or  in  the  steamer 
on  three  successive  days. 

Egg  cutiures  (Hueppe). — These  are  very  useful  for  some 
purposes.  A  hen's  egg  is  taken  and  one  end  sterilised  by 
washing  with  carbonate  of  soda  solution,  rinsing  in  sterile 
water,  soaking  in  1-500  corrosive  sublimate  solution,  and 
washing  in  alcohol  and  in  ether.  A  small  hole  is  then 
chipped  in  the  shell  with  a  sterile  needle  and  the  inocula- 
tion made  through  this.  The  hole  is  afterwards  closed 
with  a  little  sterilised  wool  and  collodion. 

Uschinsky's  Fluid.  Parts.  Pasteur's  Fluid.          Parts. 

Sodium  chloride     .          .  5-7  Cane  sugar    ...       10 

Calcium  chloride    .          .  0-1  Tartrate  of  ammonia      .         1 

Magnesium  sulphate        .  0-2-0-4  The   ash   of    1    grm.    of 

Di-potassium  phosphate  2-2-5  yeast          ...       — 

Ammonium  lactate          .  6-7  Water  ....     100 

Sodium  asparaginate       .  3-4 

Glycerin        .          .          .  30-40 

Water  ....  1000 

Uschinsky's  fluid  is  a  solution  of  known  composition  without 
protein  which  can  be  used  for  investigating  the  chemical  product 
of  bacteria.  Pathogenic  organisms  grow  well  in  it  and  produce 
their  toxins. 

Pasteur's  fluid  is  a  good  culture  medium  for  yeasts,  etc.1 

1  Several  formulae  for  synthesised  media  will  be  found  in  the  Journal 
of  Experimental  Medicine,  vol.  iii,  p.  666. 


64  A  MANUAL  OF  BACTERIOLOGY 


Standard  Nutrient  Media 

Slight  variations  in  the  composition  of  the  nutrient 
media  have  a  marked  influence  upon  the  characters  of 
the  growths  of  micro-organisms  developing  upon  them. 
In  order  to  obtain  more  uniformity  for  descriptive  pur- 
poses, etc.,  a  committee  of  the  American  Public  Health 
Association  drew  up  a  scheme  for  the  preparation  of 
nutrient  media  of  approximately  constant  composition 
and  reaction.  Eyre1  has  devoted  considerable  attention 
to  this  subject,  and  the  following  descriptions  are  based 
largely  upon  his  papers. 

(1)  Preparation  of  acid  beef -broth.- — 1000  c.c.  of  distilled 
water  are  introduced  into  a  large  flask,  500  grm.  of  finely 
minced  fresh  lean  beef  added,  and  the  mixture  is  heated 
in  a  water-bath  at  40°-45°  C.  for  twenty  minutes  with 
frequent  agitation.     It  is  then  boiled  for  ten  minutes, 
strained,    and   filtered   through   paper.     To    the    filtrate 
sufficient  distilled  water  is  added  to  make  up  to  1000  c.c. 

(2)  Standardisation. — This  may  be  most  simply  described 
in  the  case  of  acid  broth.    A  100  c.c.  Erlenmeyer  flask  is 
rinsed  out  with  boiling  distilled  water,  25  c.c.  of  the  acid 
beef-broth  are  introduced  into  it,  and  0-5  c.c.  of  phenol- 
phthalein  solution  is  added  (0-5  per  cent,  phenolphthalein  in 
50  per  cent,  alcohol).     This  is  kept  boiling  and  decinormal 
caustic  soda  solution2  is  run  in  from  a  25  c.c.  burette, 
divided  into  tenths,  until  a  faint  pink  tinge  appears  in  the 
boiling  fluid.     From  the  amount  of  soda  solution  used  the 

1  Brit.  Med.  Journ.,  1900,  vol.  ii,  p.  921  ;    1901,  vol.  ii,  p.  788. 

2  By  a  "  normal  "  solution  is  meant  the  equivalent  weight  in  grammes 
of  a  substance  dissolved  in  (i.e.  made  up  to)  a  litre  of  water ;   a  "  deci- 
normal "  solution  contains  one  tenth  of,  a  deka-normal  ten  times,  this 
amount.     A  normal  solution  of  caustic  soda  contains  40  grm.  of  pure 
NaOH    (NaOH  =  40),    of    sulphuric    acid   49    grm.    of    pure    H2S04 


STANDARD  MEDIA  65 

amount  of  normal  or  deka- normal  soda  solution  required 
to  neutralise  a  given  volume  of  the  acid  beef-broth  (e.g.  a 
litre)  can  be  calculated,  and  this  amount  is  then  added. 
Although  neutral  to  phenolphthalein,  the  medium  is  now 
strongly  alkaline  to  litmus — too  alkaline  for  the  optimum 
growth  of  most  organisms.  The  reason  for  this  is  that 
the  di-sodium  hydrogen  phosphate  (Na2HP04)  present  in 
the  medium  is  alkaline  to  litmus  but  neutral  to  phenol- 
phthalein. To  reduce  the  alkalinity  (to  litmus)  normal 
hydrochloric  acid  is  then  added.  The  American  Com- 
mittee recommended  an  acidity  of  -f-  1-5 — that  is,  to 
every  100  c.c.  of  the  medium  neutral  to  phenolphthalein 
1-5  c.c.  of  the  normal  hydrochloric  acid  are  added.  Eyre 
advises  a  reaction  of  -{-  1-0  (i.e.  1  c.c.  of  normal  hydro- 
chloric to  every  100  c.c.),  while  Chester  considers  that  the 
acidity  should  not  exceed  +  0-5.  Whatever  the  reaction 
adopted,  it  should  be  stated.  Similarly,  if  a  medium  is 
used  which  is  alkaline  to  phenolphthalein,  this  is  expressed 
by  the  minus  sign  ;  e.g.  a  reaction  of  —  1-5  indicates  that 
to  every  100  c.c.  1-5  c.c.  of  normal  hydrochloric  acid  must 
be  added  to  render  it  neutral  to  phenolphthalein,  or,  what 
is  almost  (but  not  quite)  the  same  thing,  that  to  the 
neutral  medium  1-5  c.c.  of  normal  caustic  soda  solution 
have  been  added  to  every  100  c.c.  Various  methods  are 
adopted  to  obtain  the  final  reaction ;  the  American 
Committee  recommend  first  neutralising  and  then  adding 
sufficient  acid  (or  alkali) ;  Eyre,  having  calculated  the 
acidity,  adds  only  sufficient  alkali  to  reduce  the  reaction 
to  the  required  point.  Eyre  describes  the  reaction  as  that 
represented  by  the  number  of  c.c.s  of  normal  alkali  or 
acid  per  litre,  e.g.  -f  10  on  Eyre's  scale  is  equivalent  to 
the  American  -f  1-0.  In  making  nutrient  broth,  agar 
and  gelatin,  the  salt  and  peptone  and  agar  or  gelatin  are 
added  and  dissolved,  and  the  titration  and  neutralisation 
are  carried  out  as  described,  on  the  fluid  medium  itself, 

5 


66  A  MANUAL  OF  BACTERIOLOGY 

and  after  neutralisation  the  whole  is  heated  over  a  water- 
bath  for  half  an  hour  before  filtration. 


The  Cultivation  and  Isolation  of  Micro- 
organisms 

It  should  be  clearly  understood  that  micro-organisms 
cannot  usually  be  identified  by  their  microscopical  char- 
acters alone.     We  can  state  from  a  microscopical  examina- 
tion the  form  of  an  organism,  that  it  is  a  bacillus  or  a 
micrococcus,  or  a  sarcina,  its  size,  that  it  is  motile  or  non- 
motile,  sporing  or  non-sporing,  but  we  cannot  as  a  rule 
go  beyond  this.     It  is  necessary  in  most  cases  to  ascertain 
the  characters  of  the  growths  of  organisms  on  the  various 
culture  media  before  species  can  be  identified,  and  this  is 
the  principal  reason  for  having  a  varied  assortment  of 
nutrient  soils.     It  is  likewise  necessary  for  the  successful 
cultivation  of  pathogenic  organisms,  i.e.  those  connected 
with  disease  processes  and  developing  in  or  upon  the 
bodies  of  man  and  of  animals,  to  maintain  the  cultures 
at  a  temperature  approximating  to  that  of  the  host.     For 
this  purpose  some  form  of  incubator  is  required.     This 
consists  of  a  box  or  chamber  of  copper  or  iron  with 
double  walls  (Fig.  10),  the  space  between  which  is  filled 
with  water,  the  outside  being  covered  with  wood  or  felt, 
or  some  other  non-conductor.     The  water  between  the 
walls  is  heated  by  means  of  a  small  burner,  the  gas  supply 
for  which  passes  through  some  form  of  regulator  inserted 
in  the  water,  so  that  the  temperature,  indicated  by  a 
thermometer  inserted  through  a  hole  in  the  top,  can  be 
kept  constant.     The  regulator  is  usually  a  mercurial  one, 
such  as  Page's  or  Reichert's,  the  principle  of  its  action 
being  that  as  the  temperature  rises  the  mercury  expands 
and  at  a  certain  point  cuts  off  the  greater  part  of  the  gas 
supply,  only  sufficient  gas  then  passing  to  keep  the  flame 


INCUBATORS 


'  (57 


of  the  burner  alight.  This  point  can  be  varied  either  by 
a  sliding  tube,  in  Page's,  or  by  a  screw,  in  Reichert's,  so 
that  the  temperature  may  be  set  at  any  desired  point.  In 
Hearson's  incubator,  which  is  one  of  the  best  forms,  the 


FIG.  1'). — Hearson's  incubator. 

regulator  consists  of  a  capsule  containing  a  fluid  of  a 
certain  boiling-point,  which  when  ebullition  takes  place 
raises  a  lever  and  so  partially  cuts  off  the  gas  supply. 
While  the  Hearson  regulator  is  a  very  constant  one,  it 
has  the  disadvantage  that  it  can  only  be  used  for  a  range 
of  temperature  of  a  few  degrees  unless  the  capsule  be 


68  ,  A  MANUAL  OF  BACTERIOLOGY 

changed.  At  least  one  incubator  is  required,  and  it  is 
convenient  to  have  two  or  three.  If  there  be  only  one 
the  regulator  should  be  set  for  a  temperature  of  37°  C.  ; 
if  more,  another  should  be  kept  at  about  20°  C.  The 
incubator  at  37°  C.  is  termed  the  warm  or  blood-heat, 
and  that  at  20°  C.  the  cool  or  room  temperature  one.  A 
warm  room  or  cupboard  will  serve  most  of  the  purposes 
of  the  cool  incubator.  A  third  incubator  set  for  42°  C. 
is  useful  for  water  examination,  and  a  fourth  at  25°  C.  for 
fermentation  work. 

A  substitute  for  the  large  and  expensive  incubator  can 
readily  be  devised.  An  ordinary  chemical  hot- water  oven 
may  be  employed,  or  simply  a  smaller  tin  set  in  a  some- 
what larger  one,  the  interspace  being  filled  with  water  ; 
and,  with  a  little  scheming,  regulators  can  be  dispensed 
with  by  making  use  of  a  small  gas  or  lamp  flame,  varying 
its  size  and  distance  from  the  bottom  until  the  right 
temperature  has  been  attained.  Gas  is  a  great  con- 
venience, but  if  not  available,  regulating  oil  lamps  can  be 
obtained  to  take  its  place.  Electricity  has  also  been 
adapted  for  heating  incubators. 

Gelatin  will  remain  solid  only  at  temperatures  below 
24°  C.,  and  cannot  therefore  be  placed  in  the  blood-heat 
incubator  without  becoming  for  practical  purposes  a 
fluid  medium.  Agar,  however — and  this  is  one  of  its 
most  valuable  properties — does  not  liquefy  below  a 
temperature  of  97°r99°  C.,  though  when  once  liquefied 
it  does  not  set  again  until  the  temperature  has  fallen 
to  about  45°  C.  Gelatin  is  therefore  usually  reserved 
for  use  at  low  temperatures,  while  agar,  blood-serum, 
potato,  and  the  fluid  media  can  be  used  indifferently 
either  at  low  or  at  high  temperatures.  Agar  is  often 
a  better  cultivating  medium  than  gelatin,  even  at  low 
temperatures,  probably  because  it  is  so  much  moister. 
The  growths  in  fluid  media  are  usually  of  the  nature  of  a 


INOCULATION  OF  MEDIA  69 

general  turbidity  and  are  not  particularly  characteristic, 
but  sometimes  an  organism  produces  a  film  on  the  surface 
which  another  similar  organism  does  not,  or  the  medium 
remains  clear,  the  growth  forming  a  flocculent  deposit, 
thus  affording  a  distinction.  Not  only  do  the  characters 
of  the  growths  of  organisms  on  media  differ  more  or  less, 
but  in  some  instances  chemical  changes  occur  in  the  media 
which  afford  valuable  information  in  the  differentiation 
of  species.  Thus  many  organisms  exert  a  peptonising 
effect  on  gelatin,  and  render  it  fluid  sooner  or  later,  while 
others  have  no  such  action.  Milk  is  coagulated  by  some 
organisms,  the  coagulation  being  brought  about  in  one 
of  two  ways,  either  by  the  production  of  acids  and  pre- 
cipitation of  the  caseinogen,  or  by  the  action  of  a  rennet- 
like  ferment  with  the  formation  of  a  clot  of  casein.  Most 
organisms  which  liquefy  gelatin  coagulate  milk,  but  the 
converse  is  not  the  case.  Agar  is  carbohydrate,  not 
albuminoid,  in  nature,  and  only  two  or  three  organisms 
are  known  which  liquefy  it.  In  fluid  media,  such  as 
broth  and  peptone  water,  chemical  tests  can  be  applied, 
especially  for  indole,  which  is  formed  by  some  organisms 
but  not  by  others. 

Method  of  inoculating  tubes. — The  following  is  the 
procedure  by  which  sub-cultures  are  prepared  from  an 
original  test-tube  or  other  culture  :  Tubes  of  the  culture 
media  selected  are  placed  in  a  test-tube  rack.  Their 
mouths  are  then  singed  by  holding  in  the  Bunsen  flame 
for  a  few  seconds,  and  with  a  forceps,  also  sterilised  by 
heating  in  the  flame,  the  wool  plugs  are  loosened  by  a 
rotatory  motion,  and  then  partially  withdrawn.  The 
mouth  of  the  original  culture- tube  is  similarly  singed  and 
its  plug  partially  withdrawn.  A  platinum  needle  is 
selected  and  carefully  straightened.  The  original  tube 
is  then  taken  in  the  left  hand  between  the  thumb  and 
index  finger  with  the  palm  upwards,  and  is  held  obliquely, 


70  A  MANUAL  OF  BACTERIOLOGY 

the  mouth  of  the  tube  pointing  to  the  right,  a  tube  of 
sterile  medium  being  held  side  by  side  with  the  original 
culture  in  an  exactly  similar  manner.  The  wire  of  the 
platinum  needle  is  then  heated  to  redness  by  holding 
nearly  vertically  in  the  flame,  and  the  lower  part  of  the 
handle  is  also  carefully  heated.  Holding  the  sterilised 
needle  between  the  finger  and  thumb  of  the  right  hand, 
the  plug  of  the  original  culture  is  now  withdrawn  by 
grasping  between  the  ring  and  little  fingers  of  the  right 
hand,  and  is  held  there  while  the  platinum  needle  is  care- 
fully introduced  into  the  tube  without  touching  the  mouth 
or  sides,  and  a  trace  of  the  growth  is  picked  up  with  it, 
preferably  from  the  margin.  To  ensure  that  the  needle 
is  cool,  it  may  first  be  touched  on  the  medium  where  there 
is  no  growth.  The  needle  is  quickly  withdrawn  without 
touching  the  sides  of  the  tube  and  the  plug  at  once  re- 
placed. The  plug  of  the  sterile  tube  is  now  withdrawn 
in  the  same  manner,  and  the  inoculated  needle  introduced. 
If  a  typical  surface  culture  is  desired,  a  single  light  streak 
is  made  with  the  needle  from  the  bottom  to  the  top  of  the 
medium  without  penetrating  the  surface  ;  if  an  abundant 
growth  be  required  for  any  purpose  the  whole  surface  of 
the  medium  may  be  rubbed  with  the  needle ;  if  a  stab 
culture,  the  needle  is  plunged  steadily  into  the  centre  of 
the  medium  and  withdrawn ;  if  a  fluid  one,  the  growth 
removed  is  rubbed  up  on  the  side  of  the  tube  at  the  margin 
of  the  fluid,  and  the  emulsion  washed  down  by  tilting  the 
tube.  The  inoculation  having  been  completed,  the  plug 
is  quickly  replaced,  and  the  needle  is  again  heated  in  the 
flame  to  destroy  the  remains  of  the  growth  upon  it.  If 
the  original  culture  is  in  a  deep  stab,  or  a  fluid  medium, 
a  looped  platinum  needle  may  sometimes  be  used  with 
advantage.  The  inoculations  completed,  the  mouths  of 
the  tubes  are  singed  and  the  wool  plugs  pushed  in  level 
with  the  lip.  Before  replacing  the  plugs  each  may,  if 


ANAEROBIC  CULTURES  71 

desired,  for  greater  safety,  be  taken  with  the  forceps,  held 
in  the  flame  for  a  second  or  two,  and  pushed  while 
burning  into  the  tube,  and  this  procedure  must  always 
be  adopted  if  the  plug  be  dropped  or  brush  against 
anything.  If  the  tubes  have  to  be  kept  for  any  length 
of  time,  especially  in  the  bloodheat  incubator,  each 
should  be  capped  with  a  rubber  cap,  tinfoil,  or  gutta- 
percha  tissue  which  has  been  soaked  in  1-500  corrosive 
sublimate  solution. 

Anaerobic  cultures.- — Many  organisms  refuse  to  grow  in 
the  presence  of  free  oxygen,  and  various  expedients  have 
to  be  adopted  to  exclude  or  remove  it.  The  simplest  of 
all  is  to  make  the  cultivation  in  a  deep  stab  in  glucose 
agar  or  gelatin  Narrow  test-tubes  filled  three  parts  full 
with  the  medium  are  best,  and  immediately  before  the 
inoculation  they  should  be  placed  upright  in  a  beaker  of 
water,  boiled  for  five  minutes,  and  then  cooled  and 
solidified  in  cold  water.  The  object  of  this  is  to  soften 
the  medium  so  that  it  does  not  split,  as  a  dry  medium 
will,  when  the  needle  is  plunged  into  it ;  moreover,  the 
needle  track  closes  up  more  readily,  and  the  dissolved 
oxygen  is  expelled.  The  tubes  being  cool,  the  inoculation 
is  made  with  a  long  thin  wire,  either  straight  or  with  a 
closed  loop  at  the  end.  It  is  inoculated  and  plunged 
steadily  into  the  centre  of  the  medium,  nearly  to  the 
bottom,  rotated,  and  then  withdrawn,  and  the  wool  plug 
is  replaced  and  singed.  The  tube  is  then  carefully  heated 
at  the  upper  border  of  the  medium  so  as  to  melt  this 
slightly  and  seal  the  puncture,  and  a  well- fitting  rubber  cap 
is  applied  while  the  tube  is  hot.  The  heating  expels  a 
portion  of  the  air,  and,  with  a  well-fitting  cap,  creates  a 
negative  pressure  within  the  tube,  so  that  the  residual 
oxygen  is  not  so  readily  absorbed,  or  the  tubes  may  be 
placed  in  a  Buchner  apparatus  (see  below).  The  tubes  are 
placed  in  the  incubator  at  a  suitable  temperature,  and 


72 


A  MANUAL  OF  BACTERIOLOGY 


it  will  be  found  that  the  most  strictly  anaerobic  organisms 
can  be  cultivated  in  this  way. 

When,  however,  an  organism  is  required  to  grow 
anaerobically  on  the  surface  of  the  medium,  or  in  a  fluid 
medium,  some  other  method  must  be -adopted.  The  tubes 
may  be  placed  under  the  receiver  of 
an  air-pump  and  exhausted  as  com- 
pletely as  possible.  This  is  not  very 
convenient,  for  it  is  difficult  without  great 
care  to  maintain  a  vacuum,  and  special 
receivers  must  be  used  when  the  cultures 
have  to  be  kept  in  the  incubator,  while 
with  fluid  media  ebullition  may  cause 
considerable  difficulty. 

For  fluid  cultures  Hamilton's  method 
is  the  simplest  of  all.     The  fluid  in  the 
tubes  is  covered  with  a  layer  of  olive  oil 
1-2   cm.  thick,  and  the  tubes  are   then 
sterilised.     The  layer  of  oil  prevents  the 
access  and  entrance  of  oxygen.     The  only 
disadvantage  is  that  the  inoculation,  or 
the  withdrawal  of  culture,  must  usually 
be  performed  with  a  sterile  glass  pipette ; 
FIG.  11.— Buchner's  ^  a  wire  needle  be  used  the  material  is 
tube  arranged  for  very  liable  to  be  detached  in  the  oil. 
vat^n.^  Another    method  (Buchner's)   is    that 

usually  adopted,  and  consists  in  absorb- 
ing the  oxygen  by  means  of  alkali  and  pyrogallic 
acid,  and  so  cultivating  in  an  atmosphere  of  nitrogen. 
This  can  be  carried  out  in  two  ways — either  in  a  wide- 
mouthed  bottle  with  well-fitting  glass  stopper,  sufficiently 
large  to  contain  the  test-tubes,  or  in  a  Buchner's  tube. 
For  the  first  the  inoculated  culture  tubes  are  placed 
in  the  bottle,  into  which  a  few  cubic  centimetres  of  a 
strong  aqueous  solution  of  pyrogallic  acid  have  previously 


ANAEROBIC  CULTIVATION  V\ 

been  poured.  By  means  of  a  thistle  funnel,  an  equal 
volume  of  20  per  cent,  caustic  potash1  or  soda  solution 
is  then  added.  As  quickly  as  possible  the  thistle  funnel 
is  withdrawn  without  mixing  the  solutions,  and  the  stopper, 
well  vaselined,  inserted  and  twisted  well  home,  and  some 
melted  paraffin  may  be  poured  all  round  the  joint  and 
melted  in  with  a  hot  iron.  The  solutions  in  the  bottle  are 
now  well  mixed,  and  the  whole  is  placed  in  a  suitable 
incubator.  The  Buchner's  tube  (Fig.  11)  is  convenient 
for  single  test-tube  cultures.  It  consists  of  a  strong  glass 
test-tube,  large  enough  to  take  an  ordinary  test-tube,  and 
having  a  constriction  about  an  inch  and  a  half  from  the 
bottom.  The  constriction  supports  the  test-tube  culture, 
while  the  mixture  of  pyrogallic  acid  and  .caustic  potash 
fills  the  portion  below  the  constriction.  A  well-fitting 
rubber  cork  closes  the  mouth  of  the  tube,  and  the  joint 
may  be  paraffined  for  additional  security.  If  a  Buchner's 
tube  is  not  available,  the  cotton- wool  plug  of  the  culture 
tube  may  be  pushed  into  the  tube  for  an  inch,  some  solid 
pyrogallol  is  placed  on  the  wool  plug,  this  is  just  moistened 
with  caustic  potash  solution  and  the  tube  is  stoppered 
with  a  rubber  cork. 

The  displacement  of  the  atmosphere  by  means  of 
hydrogen  may  be  adopted,  and  is  to  be  preferred  for  fluid 
cultures.  Hydrogen  does  not  seem  to  inhibit  the  growth 
of  any  anaerobic  organisms,  whereas  carbon  dioxide  gas, 
which  might  be  still  more  conveniently  used,  has  a  very 
decided  inhibitory  action  on  some  species.  The  hydrogen 
is  best  generated  from  zinc  and  sulphuric  acid  in  a  Kipp 
apparatus,  or  the  compressed  gas  in  cylinders,  or  even 
coal-gas,  may  be  used.  Care  must  be  taken  that  all 

1  Thirty-two  grm.  of  pyrogallic  acid  and  64  grin,  of  caustic  potash 
dissolved  in  100  c.c.  of  water  will  absorb  9200  c.c.  of  oxygen.  At  the 
same  time  some  carbon  monoxide  is  evolved  (122-5  c.c.).  The  evolu- 
tion of  CO  is  a  minimum  when  the  potash  is  in  excess  and  only  one- 
lifth  or  the  theoretical  absorbable  amount  of  0  is  absorbed. 


74 


A  MANUAL  OF  BACTERIOLOGY 


joints  are  tight,  and  they  may  be  paraffined  with  advan- 
tage. The  gas  should  be  passed  through  a  strong  solution 
of  caustic  potash,  and  may  be  passed  through  some 
alkaline  pyrogallic  acid  if  the  most  rigorous  condition  of 
anaerobiosis  is  desired,  but  for  ordinary  purposes  this  is 
not  essential ;  it  should  also  pass 
through  two  or  three  fairly  firm  plugs 
of  cotton- wool  to  remove  organisms  ; 
these  must  be  dry,  for  if  moist  the 
passage  of  the  gas  may  be  stopped. 

For  tube  cultures  Frankel's  method 
may  be  adopted  (Fig.  12).  The  broth 
or  gelatin  is  introduced  into  a  large 
strong  test-tube  which  is  plugged  with 
a  rubber  cork,  through  which  two 
pieces  of  glass  tubing  pass,  one  to  the 
bottom  of  the  tube,  the  other  just 
through  the  cork.  Outside  the  cork 
these  tubes  are  bent  over  at  right 
angles,  and  each  is  drawn  slightly  out 
so  as  to  contract  its  lumen  at  about 
the  middle.  The  long  tube  is  con- 
FIG.  l2.-^Frankel's  tube  nected  with  the  hydrogen  supply,  and 
for  anaerobic  cultiva-  a  current  of  the  gas  is  passed  through 
and  escapes  by  the  shorter  tube.  After 
the  gas  has  been  passing  for  twenty  minutes  to  half  an  hour, 
and  all  oxygen  has  been  expelled,  the  distal,  i.e.  shorter,  tube 
is  sealed  off  at  the  contracted  portion  in  the  Bunsen  or 
blowpipe  flame,  and  then  the  proximal  or  longer  one  in 
the  same  manner.  The  rubber  cork  must,  of  course,  fit 
well,  and  the  joints  should  be  paraffined.  If  gelatin  be 
the  medium,  it  should  be  kept  fluid  in  a  bath  of  warm 
water  while  the  hydrogen  is  passing. 

For  broth  or  other  fluid  cultures,  which  are  essential 
for  obtaining  toxic  products,  flasks  are  used  which  are 


ANAEROBIC  CULTIVATION 


75 


fitted  up  like  the  Frankel  tube  described  above.  The  ends 
of  the  tubes  are  plugged  with  cotton- wool,  and  the  whole 
— flask,  cork,  tubes  and  medium — is  sterilised.  The 
medium  is  inoculated  from  a  recent  culture  by  momentarily 
removing  the  cork.  Hydrogen  is  then  passed  through 
from  a  Kipp  apparatus,  the 
long  tube  being  connected  with 
the  hydrogen  supply.  After 
passing  for  about  half  an  hour, 
the  tubes  are  sealed  off  and  the 
flask  is  incubated.  For  con- 
venience of  sealing  the  tubes 
should  be  drawn  out  slightly. 

As  many  organisms  produce 
gas  during  their  growth,  it  may 
be  necessary  to  provide  for  its 
escape,  or  the  flasks  may  burst 
owing  to  the  pressure.  This  can 
be  done  by  adjusting  a  mercury 
valve,  and  may  be  carried  out 
in  a  simple  manner  by  a  method 
devised  by  the  writer.  "  Yeast 
flasks,"  which  can  be  obtained 
in  various  sizes,  are  made  use 
of,  and  are  filled  three  parts  full 
with  a  2  per  cent,  grape-sugar 
bouillon.  The  neck  is  corked 

With   a    perforated    rubber    cork    FIG.  13.-Yeast  flask  arranged 

for  anaerobic  cultivation. 
(A,  Fig.   13),  through    which    a 

glass  tube,  B,  passes  to  the  bottom  of  the  flask,  projecting 
two  inches  above  the  rubber  cork  and  here  plugged  with 

JT         OO 

cotton-wool.  The  lateral  tube  of  the  yeast  flask  is  also 
plugged  with  cotton- wool,  care  being  taken  that  the 
plugs  are  loose  enough  to  allow  air  to  pass  freely.  The 
whole  is  sterilised  and  inoculated.  The  glass  tube,  B, 


76  A  MANUAL  OF  BACTERIOLOGY 

which  passes  through  the  rubber  cork,  is  then  connected 
with  a  Kipp  or  other  hydrogen- genera  ting  apparatus  by 
means  of  a  rubber  tube,  and  a  current  of  hydrogen  is 
passed  through  the  flask.  The  hydrogen  bubbles  through 
the  bouillon  and  escapes  by  the  lateral  tube.  After  the 
gas  has  been  passing  for  half  an  hour  a  small  tube  con- 
taining mercury,  c,  is  applied  to  the  end  of  the  lateral 
branch,  so  that  the  open  end  just  dips  below  the  surface 
of  the  mercury,  and  the  tube,  B,  which  passes  through  the 
rubber  cork,  is  sealed  off  in  the  blowpipe  flame,  care  being 
taken  that  all  the  air  has  been  expelled  from  the  flask 
by  a  free  current  of  hydrogen.  The  flask,  with  the  capsule 
of  mercury  applied  to  the  end  of  the  lateral  branch,  can 
then  be  placed  in  the  incubator.  The  mercury  thus  forms 
a  valve  through  which  air  cannot  enter,  while  gases 
formed  by  the  growth  of  the  organism  have  free  exit. 

For  large  flasks,  the  lateral  tube  may  be  just  bent  down 
and  a  little  capsule  of  mercury  attached. 

The  addition  of  \  to  1  per  cent,  of  sodium  formate  to 
the  culture  media  much  simplifies  anaerobic  cultivation  ; 
the  tetanus  bacillus,  for  example,  can  be  grown  in  formate 
broth  in  a  stoppered  bottle  without  any  elaborate  pre- 
caution for  excluding  the  last  traces  of  air.  The  sodium 
formate  should  be  added  immediately  before  the  last 
sterilisation,  not  previously,  or  decomposition  may  occur. 
Sodium  sulphindigotate  (0-3  per  cent.)  may  be  similarly 
used. 

With  such  a  broth,  Dean's  bottle  may  be  used  for 
anaerobic  cultivation.  This  consists  of  a  bottle  around 
the  neck  of  which  a  gutter  for  mercury  is  formed.  A 
glass  cap  loosely  fits  over  the  mouth  of  the  bottle,  and  its 
edge  dips  into  the  mercury  in  the  gutter,  thus  sealing  the 
bottle. 

Plate  cultivations. — The  method  of  plate  culture  is  one 
of  the  most  important  in  bacteriology.  It  is  used  for 


PLATE  CULTIVATIONS  77 

three  purposes  :  (1)  for  obtaining  pure  cultivations,  i.e. 
cultures  containing  a  single  species,  from  a  mixture  of 
organisms  ;  (2)  for  the  enumeration  of  organisms  ;  and 
(3)  for  ascertaining  the  characters  of  the  colonies  of 
organisms  as  an  aid  in  the  identification  of  species. 

Before  the  introduction  of  plate  cultivations  pure 
cultures  of  organisms  could  only  be  obtained  by  chance, 
or  by  the  dilution  method,  which  was  also  by  no  means 
certain.  The  dilution  method  consisted  in  estimating 
approximately  the  number  of  organisms  in  a  given 
volume  of  fluid  by  means  of  an  instrument  on  the  same 
principle  as  the  hsematocytometer.  The  fluid  is  then 
diluted  by  the  addition  of  some  sterile  fluid  so  that  a 
given  volume  of  the  dilution  contains  a  single  organism 
only,  assuming  the  organisms  to  be  evenly  distributed 
throughout  the  fluid.  By  transferring  this  volume  to 
tubes  of  sterile  media  pure  cultivations  can  in  some  cases 
be  obtained,  a  single  organism  having  been  sown  in  a  tube. 

It  is  obvious,  however,  that  this  method  is  at  best  an 
uncertain  one,  but  the  plate-culture  method  to  a  large 
extent  obviates  this  uncertainty.  It  depends  upon  the 
following  principles :  Gelatin  and  agar  media,  when 
melted,  remain  fluid  down  to  25°  and  45°  C.  respectively, 
temperatures  which  will  not  affect  the  vitality  even  of 
delicate  organisms.  By  inoculating  the  fluid  gelatin  or 
agar,  thoroughly  mixing,  and  then  pouring  on  to  a  level 
sterilised  surface,  so  that  the  medium  solidifies  in  a  thin 
film  ("  plating  "),  the  organisms,  wherever  they  may  be 
situated,  are  fixed  and  are  unable  to  wander,  and,  being 
in  a  good  nutrient  soil,  grow  and  multiply  and  ultimately 
form  visible  growths  or  colonies.  Many  of  these  colonies 
will  have  arisen  from  a  single  organism  ;  the  growth, 
therefore,  is  "  pure,"  i.e.  consists  of  a  single  species,  and 
pure  cultures  can  be  obtained  by  inoculating  tubes  of 
sterile  media  from  them. 


78  A  MANUAL  OF  BACTERIOLOGY 

When  suitable,  sterile  nutrient  gelatin  is  usually 
employed  for  the  preparation  of  plate  cultivations,  as 
it  is  more  easily  manipulated  than  agar.  Three  tubes 
of  sterile  nutrient  gelatin  are  melted  at  a  low  tempera- 
ture in  a  beaker  of  water  (gelatin  melts  at  24°  C.  ;  the 
temperature  should  not  exceed  about  45°  C.).  The  tubes 
may  be  termed  respectively  1,  2,  and  3.  Tube  No.  1  is 
inoculated,  by  means  of  a  platinum  needle,  with  a  trace 
of  the  growth  from  which  pure  cultivations  are  desired.  The 

trace  of  growth  is  thoroughly 
mixed  up  and  distributed 
throughout  the  melted 
gelatin.  If  this  mixture  be 
"  plated,"  so  many  organisms 
may  be  present  in  the  film 
that  the  colonies  which  de- 

™l°P  wiH  "<*  ^  separate, 
but  will  form  a  confluent 

growth.  To  obviate  this  difficulty  a  second  and  a  third 
dilution  are  prepared.  The  second  dilution  is  made 
by  inoculating  the  tube  of  melted  gelatin  No.  2  with 
one  platinum  loopful  from  tube  No.  1,  and  thoroughly 
mixing  up ;  and  to  be  quite  sure  that  the  resulting 
colonies  will  be  isolated  from  one  another,  a  third  dilu- 
tion is  prepared  in  the  same  manner  by  inoculating  the 
tube  of  melted  gelatin  No.  3  with  two  to  four  platinum 
loopfuls  from  tube  No.  2.  The  organisms  having  been 
distributed  throughout  the  gelatin  by  rolling  and  gentle 
shaking,  the  wool  plug  is  in  each  case  withdrawn  from 
the  mouth  of  the  tube,  the  mouth  of  the  tube  is  sterilised 
in  the  Bunsen  burner  to  prevent  contamination,  then 
cooled  for  a  few  seconds,  and  finally  the  melted  gelatin  is 
poured  on  to  a  level  sterile  glass  surface.  Formerly  plates 
of  glass  were  used  (hence  the  name) ;  but  now  shallow 
glass  dishes  with  lids,  about  three  or  four  inches  in 


PLATE  CULTIVATIONS 


79 


diameter,  known  as  Petri  dishes  (Fig.  14),  are  almost 
always  employed.  They  are  previously  sterilised  in  the 
hot-air  steriliser  in  suitable  iron  or  copper  boxes  holding 
a  dozen  or  so  ;  the  melted  gelatin  having  been  poured  in, 
the  dish  is  tilted  to  diffuse  the  gelatin  over  the  bottom  of 
the  dish,  placed  on  a  level  surface  for  the  gelatin  to  set, 
and  then  stored  in  the  cool  incubator.  The  plates  are 
examined  daily,  with  a  hand  lens  if  neces- 
sary, or  with  a  low  power  of  the  micro- 
scope, the  dish  being  turned  bottom 
upwards  on  the  stage  of  the  microscope 
for  this  purpose.  When  the  colonies  have 
developed,  inoculations  can  be  made  from 
them  by  means  of  a  platinum  needle  on 
to  tubes  of  sterile  media.  The  colonies, 
having  arisen  from  single  organisms,  are 
pure,  and  the  resulting  sub-cultures  are 
therefore  also  pure  (it  sometimes  happens 
that  the  colonies  are  mixed  owing  to  two 
or  more  organisms  being  dose  together). 
Different  species  of  organisms  usually  form 
colonies  having  different  appearances,  so 
that  the  colonies  are  an  aid  in  diagnosis  and  enable  the 
various  species  to  be  picked  out  from  a  mixture.  The  colonies 
in  gelatin  are  as  a  rule  much  more  distinctive  than  those  in 
agar.  Whereas  the  plate  cultivation  prepared  from  tube 
No.  1  is  generally  too  crowded,  plates  2  or  3,  or  both,  can 
be  made  use  of,  and  it  is  apparent  that,  to  make  certain 
of  isolating  all  the  organisms  from  a  mixture,  several 
sets  of  plates  should  be  prepared.  Flat  bottles  (Fig.  15) 
may  likewise  be  used  for  plate  culturing,  and  are  also  very 
useful  for  growing  organisms  in  bulk  for  the  examination 
of  the  constituents  and  actions  of  the  bacterial  cells. 

Golding  has  devised  flat  wedge-shaped  flasks  (having 
sides  at  an  appropriate   angle)   for  plate-culturing,   and 


Fio.  15.— "Plate1 
bottle. 


80  A  MANUAL  OF  BACTERIOLOGY 

these  are  very  useful,  as  the  culture  medium  may  be  kept 
in  them  ready  for  use. 

In  addition  to  the  isolation  of  species  from  mixtures 
and  for  diagnosis,  plate  cultures  are  also  used  to  enumerate 
organisms.  Assuming  that  every  colony  arises  from  a 
single  organism,  which  is  approximately  the  case,  the 
number  of  colonies  represents  the  number  of  organisms 
originally  introduced  into  the  gelatin,  and  if  a  known 
weight  or  volume  of  the  material  inoculated  be  used,  the 
number  of  organisms  in  it  can  be  calculated.  For  example, 
in  the  bacteriological  examination  of  water  a  measured 
volume  of  the  water  is  added  to  melted  gelatin  by  means 
of  a  sterilised  pipette,  and  by  counting  the  resulting 
colonies  the  number  of  organisms  originally  present  in 
1  c.c.  of  the  water  can  be  estimated. 

Agar  plate  cultures  may  be  prepared  in  a  similar  way. 
The  agar  must,  however,  be  brought  to  a  temperature  of 
nearly  boiling  before  it  melts  ;  it  is  then  allowed  to  cool 
to  nearly  45°  C.  and  the  tubes  are  inoculated  in  the  same 
manner  as  for  a  gelatin  plate  culture  described  above. 
Unless  the  manipulations  be  carried  out  expeditiously  the 
agar  will  solidify,  or  the  agar  film  in  the  Petri  dish  be 
lumpy. 

Agar  plates  should  usually  be  inverted  during  incuba- 
tion, or  the  growth  may  become  confluent  owing  to  the 
condensation  water  carrying  the  organisms  all  over  the 
film. 

The  plate-culture  method  can  be  modified  to  suit 
particular  circumstances  :  for  example,  the  melted  gelatin 
or  agar,  uninoculated,  may  be  poured  into  the  dishes  and 
allowed  to  solidify,  and  the  film  then  inoculated  by 
streaking  or  painting  with  the  material,  or  by  pouring  a 
few  drops  of  broth  containing  the  organisms  upon  it. 
This  is  practically  the  only  way  in  which  blood-serum  can 
be  used,  the  sterile  blood-serum  being  placed  in  the  Petri 


SINGLE-CELL  CULTURES  81 

dish,  solidified  in  the  inspissator  in  the  same  manner  as 
for  blood-serum  tubes,  and  the  coagulated  film  inoculated. 

For  many  purposes  plates  are  unnecessary,  the  same 
result  being  obtained  by  rubbing  over  the  surface  of  two 
or  three  tubes  of  sloping  agar  or  gelatin  successively  the 
once  charged  needle,  straight  or  looped.  In  the  second  or 
third  tubes  isolated  colonies  generally  develop. 

The  plate-culture  method  often  fails  if  the  organism 
to  be  isolated  forms  but  a  small  minority  of  the  total 
organisms  present  in  the  mixture ;  the  only  alternative 
then  is  to  multiply  the  number  of  plates,  which,  however, 
may  entail  great  labour  in  their  examination. 

Single-cell  cultures. — With  large  cells,  such  as  yeasts, 
it  is  not  difficult  to  obtain  growths  from  single  cells  by 
making  miniature  plate  cultures  on  ruled  cover-glasses 
and  ascertaining  where  single  cells  are  located  in  the  film 
by  examining  the  preparation  with  a  J  or  £  in.  objective 
(see  Chapter  XVI).  But  with  the  minute  bacterial  cells 
this  method  is  inapplicable.  By  the  use  of  Burri's  Indian 
ink  method,1  however,  single- cell  cultures  of  bacteria  can 
be  obtained.  Fluid  Indian  ink  is  diluted  with  6-10 
volumes  of  distilled  water  and  the  mixture  is  sterilised 
in  the  autoclave.  Several  loopfuls  of  this  are  deposited 
in  series  on  a  sterile  slide.  The  first  drop  is  inoculated 
with  the  culture  which  is  being  investigated,  the  second 
drop  is  inoculated  from  the  first,  the  third  from  the  second, 
and  so  on.  A  fine  mapping-pen,  sterilised  in  the  flame, 
is  then  dipped  into  the  third,  fourth,  or  fifth  drops,  and 
the  trace  of  Indian  ink  mixture  so  picked  up  is  deposited 
on  a  gelatin  or  agar  plate.  The  droplet  is  covered  with 
a  sterilised  cover-glass  and  is  examined  with  a  J  in.  or 
J  in.  objective,  with  a  high  eyepiece.  An  organism  shows 
up  white  on  a  black  background.  Many  drops  are  de- 

1  Das  Tuschverfahren  (G.   Fischer,   1909).       Giinther  Wagner's  ink 
Hanover)  is  recommended  and  is  supplied  by  Griibler. 

6 


82  A  MANUAL  OF  BACTERIOLOGY 

posited  on  the  plate  and  examined,  and  those  in  which 
only  a  single  organism  can  be  found  are  noted  and  the  plate 
is  then  incubated  so  that  colonies  may  form,  from  which 
sub-cultures  may  be  prepared. 

Esmarctis  roll  cultures. — Another  modification  of  the 
plate-culture  method  is  known  as  Esmarch's  roll  culture. 
For  this  purpose  large  test-tubes  ("  boiling  tubes  "),  at 
least  an  inch  in  diameter  and  six  inches  long,  are  sterilised 
and  plugged  with  cotton- wool.  The  sterile  melted  gelatin, 
about  10  c.c.,  is  poured  in  and  inoculated,  the  wool  plug 
replaced,  and  the  tube  held  in  the  horizontal  position  and 
rotated  under  a  stream  of  cold  water,  or  in  warm  weather 
on  a  block  of  ice,  until  the  gelatin  has  set.  In  this  way 
the  gelatin  forms  a  thin  film  over  the  inside  of  the  tube, 
but  a  little  practice  is  required  to  get  it  evenly  distributed. 
The  colonies  then  develop  in  the  film  of  gelatin,  which  is 
quite  analogous  to  a  film  in  a  Petri  dish. 

Anaerobic  plate  cultivations  are  sometimes  required. 
The  plate  culture  after  preparation  as  described  above, 
using  a  deep  Petri  dish,  is  inverted,  and  some  alkaline 
pyrogallol  is  placed  in  the  lid ;  this  absorbs  the  oxygen 
within  the  dish.  The  preparation  must  be  kept  under 
observation  for  the  next  hour  or  so,  and  more  alkaline 
pyrogallol  is  added  from  time  to  time  to  compensate  for 
the  rise  of  fluid  within  the  dish  until  absorption  of  the 
oxygen  from  the  contained  air  is  complete. 

McLeod  has  devised  a  useful  porcelain  dish  for  con- 
taining the  alkaline  pyrogallol  over  which  the  Petri  dish 
is  inverted,  the  joint  being  made  air-tight  with  plasticine. 

In  Botkin's  method  a  bell-jar  standing  in  a  glass  dish 
is  made  use  of.  The  Petri  dishes  are  placed  on  a  support 
within  the  bell- jar,  and  mercury  or  oil  is  poured  into  the 
glass  dish.  By  means  of  a  piece  of  bent  glass  tubing  a 
stream  of  hydrogen  is  passed  into  the  bell- jar  under  its 
rini  so  as  to  displace  the  air,  which  bubbles  out  through 


FERMENTATION  TUBES  83 

the  oil  or  mercury.  When  the  air  has  been  entirely 
displaced  the  glass  tube  is  removed,  the  bell- jar  weighted, 
and  the  whole  placed  in  the  incubator.  Bulloch's  apparatus 
is  somewhat  similar  to  this.  Wide- mouthed  jars  with 
well -ground  glass  lids,  which  are  luted  down,  are  very 
convenient,  the  oxygen  being  absorbed  with  alkaline 
pyrogallol  placed  at  the  bottom,  and  the  Petri  dishes 
stacked  on  a  glass  capsule  or  other  support  to  raise  them 
above  the  fluid. 

The  Esmarch  roll  cultures  can  be  adapted  for  anaerobic 
cultures.  The  wool  plug  is  replaced  by  a  rubber  cork 
with  two  holes,  through  which  inlet  and  outlet  glass 
tubes  pass,  as  in  Frankel's  anaerobic  tubes  (Fig.  12).  The 
gelatin  (or  agar)  having  been  melted  and  inoculated,  the 
medium  is  kept  melted  in  a  water- bath  at  appropriate 
temperature,  the  hydrogen  is  passed  through  for  a  quarter 
of  an  hour,  the  tubes  are  sealed  oft',  and  the  roll- culture  is 
prepared. 

Golding's  flask  (p.  79)  or  a  "  plate "  bottle  (Fig.  15) 
may  be  similarly  used,  or  a  Golding  flask  may  be  inverted 
over  a  beaker  of  alkaline  pyrogallol. 

For  the  detection  of  fermentation  and  gas  production, 
stab  cultures  in  glucose  agar  or  shake  cultures  in  gelatin 
may  be  employed.  For  the  latter  a  tube  of  gelatin1  is 
melted  at  a  low  temperature,  inoculated  with  the  organism, 
and  allowed  to  solidify  in  the  upright  position ;  the 
organism  is  thereby  distributed  throughout  the  medium. 
Fermentation  with  gas  production  is  indicated  by  the 
presence  of  gas  bubbles,  or  even  by  the  disruption  of  the 
medium.  Durham's  fermentation  tubes  are  very  con- 
venient for  showing  fermentation.  These  are  test-tubes 
containing  suitable  fluid  media  (10  c.c.  each)  into  which 
small  glass  tubes  closed  at  the  upper  end  are  placed ;  the 

1  Lemco  gelatin  frequently  gives  no  gas ;  a  meat-broth  gelatin 
should  therefore  be  used  for  gelatin  shake  cultures. 


84  A  MANUAL  OF  BACTERIOLOGY 

latter  become  filled  during  the  sterilisation.  The  tubes 
are  inoculated  and  incubated,  and  if  fermentation  occurs 
the  little  tube  becomes  filled  with  gas  (Fig.  16).  Einhorn's 
saccharimeter  may  also  be  used  (Fig.  17).  The  tube  is 
filled  with  the  medium,  sterilised,  inoculated,  and  in- 


FIG.  16. — Durham's 
fermentation  tube. 


FIG.  17. — Einhorn's  sacchari- 
meter. 


cubated.  Any  gas  produced  collects  in  the  closed  limb 
of  the  tube.  When  the  amount  of  gas  ceases  to  increase, 
a  little  strong  caustic  potash  solution  may  be  added  ;  this 
absorbs  the  C02,  the  residue  probably  being  hydrogen, 
and  thus  the  H  :  C02  ratio  may  be  determined.  The 
most  suitable  media  for  fermentation  are  peptone  broth, 
the  acid  beef-broth  for  which  has  been  treated  with  the 


FERMENTATION  TUBES  85 

colon  bacillus  (see  p.  27),  1-2  per  cent,  peptone  water, 
or  a  medium  which  has  been  largely  used  by  Houston, 
Gordon,  and  others,  consisting  of  a  1  per  cent,  solution 
of  "  Lemco  "  in  distilled  water  with  the  addition  of  peptone 
1  per  cent.,  sodium  bicarbonate  O'l  per  cent.  ;  to  either 
medium  is  added  1-2  per  cent,  of  glucose,  lactose,  sac- 
charose, starch,  inulin,  mannitol,  dulcitol,  etc.,  and  the 
mixture  is  tinged  with  litmus. 

The  fermentation  tube  has  been  much  used  of  late  for 
the  examination  of  faeces  in  abnormal  intestinal  conditions. 
For  this  purpose  1  grm.  of  faeces  is  thoroughly  emulsified 
in  10  c.c.  of  physiological  salt  solution  and  1  c.c.  of  the 
suspension  is  introduced  into  the  fermentation  tube,  the 
long  arm  of  which  is  95  mm.  long.  The  media  employed 
are  1  per  cent,  dextrose,  lactose,  and  saccharose  broths 
made  with  "  Lemco  "  (as  above)  or  with  sugar- free  meat 
broth  (see  p.  27).  With  such  tubes  normal  stools  yield 
the  following  amounts  of  gas  : 1 

Dextrose.  Lactose.  Saccharose. 

26-75  29-9  19-5  mm. 


1  See   Herter   and   Kendall,    Studies  from   the   Rockefeller   Institute 
(Reprints),  x,  1910. 


CHAPTER  III 

THE  PREPARATION  OF  TISSUES  AND  ORGANISMS  FOR 
STAINING  AND  MOUNTING— STAINING  AND  STAINING 
METHODS 

A  SELECTED  few  of  the  numerous  methods  devised  for  the 
preparation  and  staining  of  tissues,  bacteria,  etc.,  are  here 
given.  Special  methods  occasionally  employed  will  be 
described  when  required. 

Preparation  of  Tissues 

In  bacteriological  work  the  demonstration  of  the  bacteria 
in  the  tissues  is  the  primary  object,  and,  therefore, 
the  elaborate  methods  which  have  been  devised  for  fixing 
the  tissue  elements  are  not  usually  required,  unless  it 
be  that  the  minuter  changes  in  the  latter  are  being  studied. 
The  tissues  should  always  be  obtained  as  fresh  as  possible, 
because  within  a  few  hours  of  death  they  are  invaded  by 
numerous  bacteria,  derived  from  the  air  and  from  the 
intestine,  which  may  mask  the  original  bacterial  infection 
and  lead  to  serious  mistakes  if  this  source  of  error  be  not 
carefully  borne  in  mind.  In  all  cases  the  tissue  should  be 
cut  into  pieces  of  convenient  size,  not  more  than  about 
1  cm.  in  thickness,  and  organs  if  kept  en  masse  should  be 
sliced.  Having  been  thus  prepared,  the  material  may 
be  treated  by  one  of  the  following  methods  : 

(a)  Place  directly  in 'alcohol1  for  a  week  or  a  fortnight. 

1  Methylated  spirit  may  usually  be  employed  for  all  purposes  when 
an  alcohol  of  not  more  than  90  per  cent,  strength  suffices.  // 

86 


PREPARATION  OF  TISSUES  87 

(b)  Place  in  alcohol  1  part,  water  2  parts,  for  twenty- 
four  to  forty- eight  hours,  transfer  to  alcohol  and  water, 
equal  parts,  and  finally  to  absolute  alcohol,  for  like  periods. 

(c)  Place  in  rectified  spirit  (8(5  per  cent,  alcohol)  con- 
taining 1  per  cent,  of  corrosive  sublimate  for  twelve .  to 
forty- eight  hours,  and  pass  through  increasing  strengths 
of  alcohol  as  in  (6). 

(d)  Place  for  six  to  twenty  hours  in  a  saturated  aqueous 
solution    of    corrosive    sublimate.     This    is    prepared    by 
saturating    boiling    distilled    water    with    the    corrosive 
sublimate,    cooling,    and    filtering.     Keep    in    the    dark. 
When  removed  from  the  corrosive  sublimate  solution  the 
tissues  must  be  washed  in  a  stream  of  running  water  for 
an  hour,  or,  better,  placed  for  a  day  in  70  per  cent,  alcohol 
deeply  coloured  with  iodine,   to  remove  the   excess   of 
corrosive  sublimate  and  prevent  precipitation.     The  tissues 
are  then  passed  through  increasing  strengths  of  alcohol, 
as  in  (b). 

(e)  Formalin,  a  40  per  cent,  aqueous  solution  of  formic 
aldehyde,   is   an   excellent  fixing   agent.     A   solution   of 
1  part  of  formalin  and  9  parts  of  water,  or  better,  physio- 
logical salt  solution,  may  be  used,  the  pieces  of  tissue 
remaining  in  this  for  twelve  to  twenty- four  hours.     They 
are  then  washed  in  running  water  for  an  hour  or  two  and 
passed  through  increasing  strengths  of  alcohol,  as  in  (b). 

All  tissues  after  fixing  and  hardening  should  be  pre- 
served in  alcohol — 70-80  per  cent. 

The  methods  (c),  (d),  and  (e)  are  to  be  recommended, 

however,  be  free  from  mineral  naphtha,  which  is  present  in  all  "  shop  " 
methylated  spirit.  Methylated  spirit  free  from  mineral  naphtha  can 
be  obtained  in  quantities  of  five  bulk  gallons,  "  for  scientific  purposes 
only,"  by  special  order  from  the  Inland  Revenue  Authorities,  Somerset 
House,  W.C.  If  it  cannot  be  procured,  absolute  alcohol  must  be 
employed.  Duty-free  absolute  alcohol  can  also  be  obtained  at  a  low 
price  under  somewhat  similar  conditions.  In  the  following  pages, 
when  the  unqualified  term  "  alcohol  "  is  used,  the  naphtha-free  methy- 
lated spirit  may  generally  be  employed. 


88  A  MANUAL  OF  BACTERIOLOGY 

especially  the  two  last,  as  the  tissue  elements  are  well 
fixed  thereby.  In  all  cases  the  fixing  fluid  should  be  used 
in  considerable  excess.  Fixing  fluids  containing  potassium 
bichromate  (as  in  Miiller's  fluid)  and  chromic  acid  seem 
to  prevent  the  bacteria  from  staining  with  any  certainty, 
and  should  be  avoided. 

Section  Cutting 

In  order  satisfactorily  to  demonstrate  bacteria  in  tissues, 
and  their  relation  to  the  tissue  elements,  it  is  usually 
necessary  to  prepare  sections.  For  this  purpose  either  the 
freezing  or  the  paraffin  method  should  be  employed. 

(a)  Freezing  method. — The  tissue,  in  suitable  pieces, 
must  first  be  soaked  in  water  to  remove  the  alcohol.  A 
convenient  way  of  doing  this  is  to  place  the  material  in 
a  wide- mouthed  bottle,  into  the  mouth  of  which  an 
ordinary  glass  funnel  is  introduced,  and  the  bottle  with 
the  funnel  is  placed  under  a  stream  of  running  water ; 
the  funnel,  while  allowing  the  water  to  flow  out,  retains 
the  pieces  of  tissue  in  the  bottle.  With  running  water 
the  alcohol  will  be  completely  removed  in  from  one  to 
two  hours ;  in  still  water,  which  should  be  changed  two 
or  three  times,  this  result  may  not  be  attained  for  several 
hours,  during  which  time  there  is  an  ever-increasing  risk 
of  bacterial  contamination  from  without.  It  is  essential 
to  remove  all  the  alcohol,  or  the  tissue  will  not  freeze. 

When  the  alcohol  has  been  removed,  which  is  known 
by  the  tissue  sinking  in  the  water  (lung  is  an  exception 
— it  always  floats  unless  solid  from  any  cause),  the  pieces 
are  transferred  to  a  strong  mucilage  of  gum  acacia  : 

Gum  acacia  ......          5  grm. 

Cane  sugar  .          .          .          .          .          .       0-5  grm. 

Water 100  c.c. 

Add  a  piece  of  thymol  or  a  little  carbolic  acid  to  prevent  decom- 
position. Hamilton  saturates  the  solution  with  boric  acid. 


SECTION  CUTTING  89 

In  this  gum  solution  the  pieces  remain  for  twelve  to 
forty- eight  hours,  according  to  their  size  and  the  time 
at  the  disposal  of  the  investigator,  and  are  then  cut  on 
one  of  the  numerous  ether-freezing  microtomes  now  to  be 
obtained,  such  as  Swift's  (Fig.  18)  or  Cathcart's.  A 


FIG.  18. — Swift's  ether-freezing  microtome. 

microtome  in  which  the  freezing  is  effected  by  carbonic 
acid  is  now  frequently  employed  and  acts  well.  Liquid 
carbonic  acid,  contained  in  a  cylinder,  sprays  by  its  own 
pressure  on  to  the  under  surface  of  the  plate  on  which  the 
block  of  tissue  rests ;  the  tissue  quickly  freezes  and  is 
then  cut.  This  form  of  microtome  works  satisfactorily  in 
the  hottest  weather.  The  material  must  not  be  frozen  so 
hard  that  the  sections  roll  up  and  fall  off  the  knife ;  the 
sugar  in  the  above  solution  should  prevent  this.  The 
sections  are  transferred  successively  to  two  or  three  lots 


90  A  MANUAL  OF  BACTERIOLOGY 

of  distilled  water,  preferably  slightly  warmed,  to  remove 
the  gum,  and  can  then  be  stained  at  once,  or  may  be 
preserved  in  equal  parts  of  alcohol  and  water. 

Bacteria  seem  to  retain  their  staining  properties  better 
in  the  tissue  in  bulk  than  in  sections  preserved  in  alcohol. 
This  objection  does  not  hold  with  paraffin  sections. 

(b)  Paraffin  method. — Nothing  can  surpass  the  paraffin 
method  for  the  thinness  and  beauty  of  the  sections  obtain- 
able by  it,  and  for  some  friable  tissues,  such  as  actino- 
mycosis,  it  is  almost  essential.  The  tissue,  in  suitable 
pieces  for  cutting,  is  transferred  from  the  diluted  spirit 
preservative  solution  to  pure  methylated  spirit  for  two 
or  three  hours,  and  then  to  absolute  alcohol — which  may 
have  to  be  changed  once  unless  a  fairly  large  volume  is 
employed — for  from  four  to  twenty- four  hours.  It  is 
then  removed  from  the  alcohol,  lightly  dried  between  the 
folds  of  a  dry  cloth  or  piece  of  blotting-paper  to  remove 
the  superfluous  alcohol,  and  placed  in  an  excess  of  xylol, 
in  which  it  remains  for  from  four  to  twenty-four  hours 
until  cleared.  This  is  recognised  by  the  material  assuming 
a  more  or  less  semi-transparent  condition,  and  the  pro- 
cess may  be  much  accelerated  by  warming  the  xylol  to 
from  37°  to  50°  C.  in  the  blood-heat  incubator  or  paraffin 
oven,  the  bottle  containing  the  xylol  being  well  stoppered. 
When  cleared  it  is  ready  to  go  into  the  bath  of  melted 
paraffin.  A  paraffin  of  a  fairly  high  melting-point  is 
perhaps  the  best,  viz.  45°  to  55°  C.,  and  is  placed  in  glass 
capsules  in  an  oven  which  can  be  kept  uniformly  heated 
to  the  required  temperature.  An  ordinary  chemical  hot- 
water  oven  answers  the  purpose  quite  well,  and  is  heated 
by  a  special  form  of  small  Bunsen  burner  with  mica 
chimney,  the  temperature  being  regulated  by  some  form 
of  mercurial  regulator,  which  is  set  a  degree  or  two  above 
the  melting-point  of  the  paraffin  employed.  The  tissue 
is  taken  out  of  the  xylol,  blotted  to  remove  the  excess,  and 


PARAFFIN  SECTIONS  91 

placed  in  the  melted  paraffin  for  from  six  to  twenty  hours. 
It  is  then  embedded  by  pouring  a  little  of  the  melted 
paraffin  into  a  watch-glass,  or  into  a  small  box  formed 
of  folded  paper  or  lead-foil,  or  by  bringing  together 
two  L- shaped  pieces  of  brass  on  a  glass  plate  so  that  a 
rectangular  cavity  is  produced.  The  pieces  of  tissue  are 
then  taken  out  with  a  small  warmed  forceps  or  needle, 
adjusted  to  the  position  they  are  required  to  occupy, 
and  more  melted  paraffin  is  poured  in,  so  as  to  cover  them. 
When  a  film  of  solid  paraffin  has  formed,  the  whole  is 
immersed  in  cold  water  so  as  to  cool  it  rapidly. 

A  new  paraffin  is  frequently  crystalline  in  structure, 
and  acts  much  better  after  it  has  been  kept  melted  for 
some  weeks,  or  is  much  improved  by  heating  nearly  to 
its  boiling-point  for  five  or  six  days  (P.  T.  Beale).  The 
xylol  for  clearing  may  be  used  several  times  and  the 
paraffin  repeatedly,  the  remains  of  old  tissues  being 
removed.  The  time  which  the  tissues  require  to  remain 
in  the  alcohol,  xylol,  and  paraffin  depends  upon  their 
size  ;  very  small  pieces  may  be  treated  in  a  few  hours, 
large  ones  may  require  two  or  three  days. 

Other  clearing  agents,  such  as  chloroform,  turpentine, 
and  cedar  oil,  may  be  used  instead  of  xylol.  The  paraffin 
method  is  usually  straightforward,  but  small  pieces  of 
tissue  must  not  be  left  too  long  either  in  absolute  alcohol 
or  in  the  paraffin  bath,  for  they  are  liable  to  become  too 
hard  to  cut.  Thyroid  tissue  and  skin  are  also  rather 
troublesome  ;  they  become  very  hard  unless  the  whole 
process  is  carried  out  as  rapidly  as  possible.  If  the  pieces 
of  tissue  be  large,  the  capsule  of  melted  paraffin  containing 
the  tissue  may  be  placed  under  the  receiver  of  an  air- 
pump,  which  is  then  exhausted.  This  causes  the  paraffin 
to  penetrate  better,  and  the  process  may  be  repeated  two 
or  three  times  during  the  period  of  infiltration.  A  special 
form  of  paraffin  oven  has  been  devised  by  Cheatle  for 


92  A  MANUAL  OF  BACTERIOLOGY 

infiltrating  under  diminished  pressure,  and  is  made  by 
Messrs.  Hearson,  of  Regent  Street,  London. 

In  order  to  prepare  sections  from  material  embedded  in 
paraffin  some  form  of  microtome  must  be  employed.  An 
ether-freezing  microtome  can  be  made  use  of  with  some 
manipulation,  the  paraffin  block  being  placed  in  a  little 
melted  paraffin  on  the  freezing  plate  so  that  it  is  cemented 


FIG.  19. — Cambridge  rocking  microtome. 

there,  and  sections  are  cut  with  the  razor  or  plane  iron, 
as  though  it  had  been  frozen  (it  is  not  to  be  frozen).  It  is 
better,  however,  to  use  some  special  form  of  microtome, 
the  Cambridge  "  Rocker  "  (Fig.  19),  or  a  modification  of 
it,  or  the  "  Minot,"  being  perhaps  the  best.  The  block  of 
paraffin  containing  the  tissue  is  trimmed  with  a  knife  to 
remove  the  excess,  and  is  cemented  to  the  carrier  of  the 
microtome  with  a  little  melted  paraffin,  or  by  melting 
the  paraffin  on  it  with  a  hot  iron  (end  of  a  file,  etc.)  or  a 
match.  The  union  may  be  made  more  secure  by  melting 
the  paraffin  around  the  base  of  the  block  with  a  hot  iron. 
Having  fixed  the  paraffin  block  to  the  carrier,  sections 
may  then  be  cut  of  any  degree  of  thinness.  In  order  to 


MICROTOMES  93 

do  this  it  is  essential  for  the  knife  or  razor  to  have  a  keen 
edge  and  one  of  the  right  nature,  for  a  knife  may  be 
perfectly  sharp  and  yet  the  sections  as  they  are  cut  may 
roll  up  in  such  a  manner  that  it  is  difficult  to  flatten  them. 
Though  this  may  be  due  to  a  wrong  consistence  of  the 
paraffin,  owing  to  cold  weather  or  some  other  factor,  in 
the  majority  of  instances  it  is  the  edge  of  the  knife  which 
is  at  fault.  Provided  the  knife  be  sharp,  stropping  on 
the  palm  of  the  hand  will  usually  remedy  this  difficulty. 
The  paraffin  being  of  the  right  consistence,  and  the  knife 
in  good  order,  the  sections  as  they  are  cut  should  be  flat 
and  should  adhere  together  at  adjacent  margins  so  that 
a  ribbon  of  greater  or  shorter  length  is  formed. 

Satisfactory  sections  having  been  obtained,  they  are 
transferred  with  a  needle  or  camel's-hair  brush  to  a  tin 
pan  containing  a  little  water,  or  spirit  and  water  warmed 
to  about  40°  C.  The  sections  float  and  the  paraffin  softens 
so  that  they  spread  out  perfectly  flat  (the  water  must 
not  be  hot  enough  to  melt  the  paraffin).  A  clean  slide  is 
then  introduced  underneath  the  section,  raised  so  that 
the  section  is  lifted  up  on  it,  and  by  fixing  the  section  with 
a  needle  and  tilting  the  slide  the  section  is  deposited  in 
the  required  position  on  the  slide  and  allowed  to  dry. 
If  preferred,  the  section  may  be  transferred  to  a  slide 
flooded  with  water,  which  is  warmed  over  the  Bunsen. 
The  slides  can  be  manipulated  in  an  hour  or  two  if  dried 
at  37°  C.,  but  it  is  best  to  allow  them  to  dry  in  the  incubator 
all  night.  It  will  be  found  after  this  treatment  that  thin 
sections  generally  adhere  sufficiently  firmly  to  the  slides 
for  all  the  ordinary  methods  of  staining  to  be  carried  out 
without  detaching  them ;  thick  sections,  however,  do  not 
adhere  nearly  so  well. 

To  prevent  the  risk  of  detachment,  it  is  generally  better 
to  cement  the  sections  to  the  slides  by  the  following 
method.,  Equal  parts  of  egg-white  and  glycerin  are  mixed 


94  A  MANUAL  OF  BACTERIOLOGY 

and  filtered  through  muslin,  and  to  every  100  c.c.  of  the 
mixture  1  grm.  of  sodium  salicylate  is  added.  The  slide 
is  smeared  thinly  with  this,  the  section  is  transferred  to  it 
and  afterwards  dried  in  the  manner  above  described. 

Supposing  that  the  sections,  in  spite  of  all  precautions, 
curl  up  as  they  are  cut,  it  is  still  often  possible  to  obtain 
a  few  that  can  be  mounted.  They  may  sometimes  be 
unrolled  by  cautious  manipulation  with  a  couple  of 
needles  after  having  been  softened  by  warming,  or  a 
needle  or  knife- blade  may  be  held  close  to  the  edge  of 
the  microtome  knife  during  cutting,  so  that  curling  is 
prevented. 

Tissues  embedded  in  paraffin  may  be  kept  indefinitely  in  labelled 
pill-boxes  and  cut  all  at  once  or  from  time  to  time  as  required,  or 
the  ribbons  of  sections  may  be  preserved  in  a  box  in  a  cool  place 
until  wanted.  The  slides  also,  with  the  sections  attached,  can  be 
kept  until  it  is  convenient  to  stain,  if  preserved  free  from  dust  in 
a  slide  box. 

Cover-glass  and  Film  Specimens 

The  satisfactory  preparation  of  cover- glass  and  film 
specimens  is  one  of  the  most  important  in  bacteriology, 
for  they  are  used  for  the  examination  of  cultivations  of 
bacteria,  and  of  blood  or  other  fluids  or  secretions,  organs, 
etc.,  for  the  presence  of  micro-organisms. 

Films  and  smears  are  now  usually  made  on  the  slide, 
but  may  be  made  on  the  cover- glass  ("  cover- glass  speci- 
mens ").  In  either  case  the  glass  must  be  clean  and  free 
from  grease.  Cover- glasses  must  be  thin,  otherwise  the 
higher  powers  cannot  be  employed  to  examine  the  prepara- 
tions, and  those  described  as  "  No.  1  "  should  be  purchased, 
"  f-in.  squares  "  being  a  convenient  size.  These  serve 
both  for  cover- glass  specimens  and  for  covering  sections  ; 
it  is  well  also  to  have  a  few  of  the  same  thickness  but 
larger,  viz.  |-in.  or  1-in.  squares,  for  large  sections. 


FILM  PREPARATIONS  95 

Slides  and  cover-glasses  may  be  cleaned  by  boiling  them 
in  a  porcelain  dish  with  10  per  cent,  carbonate  of  soda 
solution  for  a  few  minutes,  well  washing,  and  then  treating 
with  strong  sulphuric  acid,  warmed  carefully  in  a  porcelain 
dish,  for  a  few  minutes.  The  acid  having  been  poured  off, 
they  are  well  rinsed  in  several  changes  of  water,  and 
should  be  kept  in  a  stoppered  glass  pot  or  capsule  in 
absolute  alcohol. 

A  clean  slide  (or  cover- glass)  is  taken,  dried  with  a 
clean  soft  linen  or  silk  rag  or  handkerchief,  or  with 
Japanese  paper,  or  it  may  be  momentarily  introduced  into 
the  Bunsen  flame  and  the  spirit  burnt  off,  and  placed 
flat  on  a  convenient  support  on  the  work-table — a  white 
glazed  tile  is  excellent — with  the  end  or  corner  projecting 
so  that  it  can  be  conveniently  picked  up.1  A  droplet 
(i.e.  small  drop)  of  tap-water  or  of  physiological  salt 
solution  (not  distilled  water)  is  then  placed  on  it,  in  the 
middle,  by  means  of  a  looped  platinum  needle,  or  with 
a  small  glass  pipette  (Fig.  7).  Theoretically,  physiological 
salt  solution2  sterilised  by  boiling  should  be  used,  but 
ordinary  tap- water  may  generally  be  employed.  A  thin 
film  of  organisms  has  now  to  be  formed  on  the  glass,  and 
the  following  is  the  method  of  procedure  with  a  culture 
on  a  solid  medium  such  as  agar  or  gelatin.  The  culture 
tube  and  platinum  needle  are  held  and  manipulated  in 
precisely  the  same  manner  as  that  described  for  the 
inoculation  of  tubes  (p.  69). 

A  mere  trace  of  the  growth  from  a  culture  should  be 
taken,  just  sufficient  to  soil  the  tip  of  the  straight  platinum 
needle,  or  the  preparation  will  be  too  crowded,  and  this 
is  well  rubbed  up  with  the  droplet  of  water  on  the  glass, 

1  The  writer  has  devised  a  useful  support  for  staining.     It  consists 
of  a  square  of  plate  glass,  painted  half  white  and  half  black  at  the 
back,  and  having  a  narrow  strip  of  thick  glass  cemented  across  it  on 
which  the  gl":js  rests.     It  is  made  by  Messrs.  Baird  and  Tatlock. 

2  0-75-0-95  per  cent,  of  sodium  chloride  dissolved  in  distilled  water. 


96  A  MANUAL  OF  BACTERIOLOGY 

so  as  to  form  an  emulsion,  which  is  then  spread  over  the 
surface.  As  a  general  rule  the  material  should  be  well 
emulsified,  but  in  some  instances  this  is  inadvisable,  as  a 
particular  formation  or  characteristic  grouping  may  be 
disturbed  thereby,  in  which  case,  after  a  slight  admixture 
with  the  water,  the  emulsion  is  gently  spread.  The  thin 
moist  film  is  allowed  to  dry,  or  may  be  dried  by  gentle 
warming  over  the  Bunsen  flame,  preferably  holding  the 
preparation  in  the  fingers  and  moving  backwards  and 
forwards  over  the  flame.  The  film,  when  dry,  must  next 
be  fixed,  which  is  accomplished  by  passing  the  slide, 
film  side  up,  six  times  through  the  Bunsen  flame  (a  cover- 
glass  is  held  in  the  forceps  and  passed  three  times  through 
the  flame).  Films  may  also  be  fixed  in  alcohol  and  ether 
(p.  97).  The  object  of  this  "  fixing "  is  to  thoroughly 
dry  the  film  and  coagulate  albuminous  material,  whereby 
the  film  adheres  better  to  the  glass,  and  is  not  so  likely 
to  be  detached  in  the  subsequent  processes  of  staining 
and  washing,  etc.  Fixing  may  also  tend  to  diminish  the 
staining  capacity  of  the  extraneous  matter  mixed  with 
the  organisms.  The  preparations  are  now  ready  for 
staining. 

When  the  culture  is  in  a  fluid  medium,  such  as  broth, 
the  tube  is  manipulated  in  the  same  way,  the  deposit  at 
the  bottom  having  been  shaken  up  if  necessary,  and  a 
loopful  or  two  of  the  fluid  removed  with  a  looped  platinum 
needle,  transferred  to  the  glass,  spread,  dried,  and  fixed 
as  before,  but  as  the  medium  is  fluid  there  is  usually  no 
need  to  add  any  water. 

If  a  specimen  of  blood,  pus,  or  sputum  is  required,  the 
procedure  is  much  the  same.  A  little  of  the  material  is 
taken  up  with  a  looped  platinum  needle  and  spread  in  a 
thin  film  over  the  slide  or  cover- glass,  which  is  then  dried 
and  fixed,  as  described  above.  If  necessary,  a  droplet 
of  tap  water  or  physiological  salt  solution  may  be  used 


SMEAR  PREPARATIONS  97 

to  dilute  the  material  so  as  to  obtain  a  thinner  film.  If  a 
specimen  is  to  be  made  from  an  organ,  a  particle  of  the 
pulp  is  picked  up  and  an  emulsion  made  as  before,  or  a 
small  piece  of  the  organ  may  be  held  in  sterile  forceps  and 
the  cut  surface  gently  smeared  over  the  slide  or  cover- 
glass,  which  is  then  similarly  dried  and  fixed  ;  these  are 
termed  "  smear  preparations." 

To  obtain  the  best  results  it  is  preferable  before  staining 
to  submit  films  of  blood1  or  pus  or  smear  preparations  to 
the  action  of  some  chemical  fixing  agent,  unless  the  film 
is  stained  with  Leishman's  solution,  which  both  fixes  and 
stains.  The  simplest  method  of  doing  this  is  to  immerse 
the  films,  after  owr-drying,  in  a  mixture  of  equal  parts  of 
absolute  alcohol  and  ether  for  ten  to  thirty  minutes.  In 
hot  countries  a  saturated  aqueous  solution  of  corrosive 
sublimate  (five  to  fifteen  minutes)  is  perhaps  as  satisfactory 
as  anything.  Another  method,  combining  both  fixing  and 
staining,  is  to  immerse  the  films  as  soon  as  they  are  pre- 
pared and  without  drying  for  a  few  minutes  in  the  following 
solution  : 

Absolute  alcohol,  saturated  with  eosin         .          .  25  c.c. 

Pure  ether       .......  25  c.c. 

Alcoholic  solution  of  corrosive  sublimate  (2  grm. 

in  10  c.c.)     .......  5  drops 

The  specimens  are  then  removed  with  a  forceps  and  well 
rinsed  in  water,  stained  for  not  more  than  a  minute  in  a 
saturated  aqueous  solution  of  methylene  blue,  washed 
quickly,  dehydrated  in  absolute  alcohol,  cleared  in  xylol, 
and  mounted  in  xylol  balsam.  This  solution  may  be 
used  for  fixing  blood,  pus,  sputum,  etc.,  if  the  eosin  be 
omitted,  and  the  preparations  may  then  be  stained  or 
otherwise  treated  in  any  desired  manner.2 

1  For  the  method  of  preparing  blood-films  see  the  section  on  "  Ma- 
laria," Chapter  XVIII. 

2  Gulland,  Brit.  Med.  Journ.,  1897,  vol.  i,  p.  65. 

7 


98  A  MANUAL  OF  BACTERIOLOGY 

Scott1  recommends  the  following  as  giving  the  most 
perfect  results  with  blood  films,  etc.  : 

(1)  Hold  the  freshly  prepared  and  still  wet  film  in  the 
mouth   of   a   wide-mouthed   bottle   half   filled   with   the 
ordinary    formalin  solution,  film  side  downwards,  for  five 
seconds. 

(2)  Drop,  while  still  wet,  film  downwards,  into  absolute 
alcohol.     Leave  for  fifteen  minutes,  or,  for  convenience, 
for  any  time  up  to  forty- eight  hours. 

The  preparations  may  then  be  stained  with  methylene 
blue,  hsematoxylin  and  eosin,  or  with  the  Leishman  or 
Giemsa  stain.  (See  also  under  "  Malaria,"  Chapter  XVIII.) 

Impression  specimens. — These  are  employed  to  examine 
and  preserve  permanently  the  colonies  or  growths  of 
organisms  so  that  their  characteristic  formation  may  be 
observed.  With  plate  cultivations  this  is  very  simple. 
A  clean  cover-glass  is  sterilised  in  the  flame  and,  having 
cooled,  is  cautiously  lowered  on  to  a  selected  surface 
colony  with  a  sterile  needle,  avoiding  all  lateral  movement. 
It  is  then  gently  pressed  on  to  the  colony  and  then  care- 
fully raised  by  means  of  a  couple  of  needles  ;  the  colony 
should  adhere  to  the  glass,  and  may  be  dried  and  fixed. 
The  colonies  in  gelatin  tube  cultures  may  also  be  used  if 
the  gelatin  is  removed  from  the  tube.  This  can  be  done 
by  dipping  the  tube  for  a  few  seconds  into  hot  water  ; 
the  gelatin  round  the  walls  of  the  tube  will  be  melted, 
and  the  gelatin  mass  can  then  be  tilted  out  of  the  tube 
on  to  a  glass  dish  or  tile. 

Stains  and  Staining  Methods 

Micro-organisms  being  so  minute  and  transparent,  it 
is  usual  to  stain  or  dye  them,  so  that  they  can  be  more 
readily  examined.  In  some  instances  organisms  may  have 

1  Journ.  Path,  and  Bact.,  vol.  vii,  No.  1,  p.  131. 


STAINS  AND  STAINING  METHODS  99 

a  peculiar  staining  reaction  which  may  serve  as  an  aid  to 
their  identification.  But  when  an  organism  is  being 
investigated,  examination  in  the  fresh  and  living  condition 
must  never  be  omitted,  for  it  is  only  thus  that  its  motility 
and  life-history  can  be  studied.  Only  general  methods 
are  detailed  here ;  special  ones  will  be  given  when  they 
are  required. 

(1)  LofHer's  alkaline  methylene  blue  : 

Saturated  alcoholic  solution  of  methylene  blue  .       30  c.c. 
Solution  of  caustic  potash,  0-01  per  cent.  .          .     100  c.c. 

A  very  useful  staining  solution.  Cultures  should  be  quite  fresh, 
or  the  organisms  do  not  stain  well.  When  the  organisms  are 
mixed  with  extraneous  material,  as  in  smears,  or  there  is  much 
debris,  this  is  one  of  the  best  staining  solutions  to  employ.  Methyl- 
ene blue  preparations  are,  however,  not  very  permanent,  and  in 
hot  countries  rapidly  fade.  Thionine  blue  is  then  preferable.  (See 
also  p.  100.) 

Film  specimens  are  stained  for  three  to  ten  minutes,  and 
sections  half  to  twenty-four  hours. 

(2)  Carbol-methylene  blue  (Kiihne) : 

Methylene  blue       .          .          .          .          .          .1-5  grin. 

Absolute  alcohol     .          .          .          .          .  10  c.c. 

Five  per  cent,  aqueous  solution  of  carbolic  acid  .     100  c.c. 

A  more  intense  staining  solution  than  the  former,  and  very 
useful  for  sections,  which  are  stained  for  from  half  to  six  hours. 

(3)  Anilin  gentian  violet : 

Saturated  alcoholic  solution  of  gentian  violet     .       30  c.c. 
Anilin  water  .......     100  c.c. 

The  anilin  water  is  prepared  by  shaking  3  c.c.  of  anilin  with 
90  c.c.  of  distilled  water,  allowing  the  mixture  to  stand  for  a  few 
minutes,  and  filtering. 

This  solution  is  a  useful  general  stain  for  films,  which  are  stained 
for  two  or  three  minutes,  and  is  employed  in  Gram's  method  of 
staining.  It  does  not  keep  well. 

Instead  of  anilin  gentian  violet,  a  carbol-gentian  violet  may  be 
used,  and  keeps  much  better  than  the  foregoing  (saturated  alcoholic 


100  A  MANUAL  OF  BACTERIOLOGY 

solution  of  gentian  violet,  1  part ;   5  per  cent,  aqueous  solution  of 
carbolic  acid,  10  parts). 

For  anilin  gentian  violet  two  stock  solutions  may  be  employed, 
and  these  seem  to  keep  indefinitely,  viz.  : 

No.  1 
Gentian  violet         ......         2  grm. 

Anilin   ........         9  c.c. 

Alcohol  (95  per  cent.)      .....  33  c.c. 

No.  2 
Gentian  violet         ......         2  grm. 

Distilled  water 100  c.c. 

For  use,  mix  1  c.c.  of  No.  1  with  9  c.c.  of  No.  2,  and  filter  ;  this 
mixture  will  keep  for  about  a  fortnight. 

(4)  Carbol-fuchsin  (Ziehl-Neelsen  solution) : 

Fuchsin          .......         1  part 

Absolute  alcohol     .          .          .          .          .    •       .       10  parts 

Five  per  cent,  aqueous  solution  of  carbolic  acid  .     100  parts 

The  fuchsin  is  dissolved  in  the  absolute  alcohol  and  then  mixed 
with  the  carbolic  acid  solution.  It  must  always  be  filtered  before 
use. 

An  intense  staining  solution.  For  films  it  is  best  diluted  with 
five  to  ten  parts  of  water  ;  stain  for  two  to  five  minutes. 

(5)  Carbol-thionine  blue  (Nicolle) : 

Saturated  solution  of  thionine  blue  in  alcohol 

(90  per  cent.) 10  c.c. 

One  per  cent,  aqueous  solution  of  carbolic  acid    .     100  c.c. 

Sections  can  be  stained  in  from  a  few  minutes  to  half  an  hour. 
This  solution  may  be  used  for  a  modified  Gram's  method  (see 
p.  106).  Can  be  substituted  for  methylene  blue  for  all  purposes, 
and  is  more  permanent  than  the  latter. 

(6)  Eosin  (alcohol-soluble  and  water-soluble) : 

A  somewhat  diffuse  stain.  Is  used  for  counter-staining  the 
tissues  in  Gram's  method,  and  for  staining  red  blood-corpuscles 
and  acidophile  granules  in  leucocytes. 

A  |  to  1  per  cent,  aqueous  or  alcoholic  solution  may  be  used, 
and  the  staining  should  not,  as  a  rule,  be  prolonged  for  more  than 
about  half  a  minute. 


STAINING  SOLUTIONS  101 

(7)  Bismarck  brown  (Vesuvin) : 

A  saturated  aqueous  solution  should  be  prepared  and  diluted 
somewhat  for  use.  A  good  counter-stain  for  the  tissues  in  Gram's 
method.  Stain  for  two  to  five  minutes. 

(8)  Orange-rubin  : 

Prepare  saturated  aqueous  solutions  of  orange  G.  and  rubin  S. 
Mix  equal  volumes  and  dilute  with  water  until  of  a  light  port-wine 
colour.  Stain  tissues  for  five  to  fifteen  minutes.  A  good  contrast 
stain  for  tuberculosis  and  actinomycosis. 

(9)  Picro-carmine : 

This  is  best  bought  ready  prepared.  Sections  are  stained  in  the 
solution  for  half  to  one  hour,  washed,  then  placed  in  a  watch-glass 
of  spirit,  to  which  three  or  four  drops  of  hydrochloric  acid  have 
been  added,  for  two  or  three  minutes,  then  well  washed  in  water. 
The  section  can  now  be  counter-stained  with  Loffler's  blue  or  by 
Gram's  method. 

(10)  HaBmatoxylin  : 

Ehrlich's  formula  is  one  of  the  best  and  simplest  to  use,  and 
can  be  obtained  ready  for  use.  It  must  be  "  ripe."  It  is  a  histo- 
logical  and  not  a  bacterial  stain.  Sections  are  treated  as  follows  : 

(1)  Distilled  water,  one  to  two  minutes. 

(2)  Stain  with  the  hsematoxylin  solution  for  five  to  thirty  minutes. 
In  some  cases  the  solution  is  preferably  diluted  somewhat  with 
distilled  water. 

(3)  Rinse  in  distilled  water. 

(4)  Rinse  in  distilled  water  containing  a  trace  of  acetic  acid. 

(5)  Treat  with  distilled  water  containing  a  trace  of  ammonia. 
The  sections  remain  in  this  until  they  assume  a  deep  blue  colour. 
(Tap-water,  five  to  ten  minutes,  may  also  be  used.) 

(6)  They  can  be  dehydrated,  cleared  and  mounted,  or  counter- 
stained  with  eosin,  orange-rubin,  or  Van-Gieson,  and  then  mounted. 

Hsematoxylin  makes  a  good  contrast  stain  for  the  tubercle  and 
the  leprosy  bacillus  and  for  Actinomyces. 

Mayer's  hsemalum  (see  section  on  the  "  Amoeba  coli ")  and 
Delafield's  hsematoxylin  are  also  good  hsematoxylin  stains. 

(11)  Ehrlich-Biondi  triple  stain  : 

This  is  best  bought  ready  for  use.     It  is  a  good  histological  stain 
for  tissues  and  blood  films,  and  actinomycosis  stains  well  by  it. 
Stain  for  ten  to  sixty  minutes,  then  treat  with  methylated  spirit 


102  A  MANUAL  OF  BACTERIOLOGY 

until  the  section  becomes  greenish.     Pass  through  absolute  alcohol, 
clear,  and  mount. 

(12)  Leishman's  stain : 

Like  the  Jenner,  Wright,  and  other  similar  ones,  a  modification 
of  the  Romanowsky  stain,  a  double  compound  of  eosin  and  methyl  - 
ene  blue.  The  solution  will  keep  for  some  time,  but  is  best  freshly 
prepared.  Griibler's  powder  or  Burroughs  Wellcome 's  soloid 
may  be  used,  and  is  dissolved  in  pure  (Merck's  or  Kahlbaum's) 
methyl  alcohol.  Failure  frequently  proceeds  from  the  use  of  a 
so-called  pure  methyl  alcohol,  which  is  not  really  so.  (For  method 
of  using,  see  "  Malaria,"  Chapter  XVIII.) 

(13)  Giemsa  stain: 

An  eosin-azur  mixture  dissolved  in  pure  glycerin  and  methyl 
alcohol.  Useful  for  blood-films,  smears,  etc.,  and  has  been  much 
used  to  demonstrate  the  spirochaetes  in  syphilitic  material.  (For 
method  of  using,  see  "  Syphilis  "  and  "Malaria.") 

Safranin  and  acid  fuchsin  are  also  used  as  counter-stains. 
Malachite  green,  neutral  red,  and  rosein  may  be  used  for  intra-vitam 
staining  of  protozoa,  etc. 

Eosin,  orange-rubin,  hsematoxylin,  and  picro-carmine  keep  well 
in  solution  ;  the  remainder  may  or  may  not,  and  are  best  used 
fairly  fresh.  All  stains  should  be  filtered  before  use,  and  may  be 
conveniently  kept  in  bottles  having  a  funnel  fitted  with  a  filter- 
paper,  so  that  they  are  always  ready.  Or  smaller  bottles  may  be 
used,  fitted  with  pipettes,  and  several  arranged  in  a  stand. 

Methylene-blue,  Leishman  and  Giemsa  preparations  are  more 
permanent  if  kept  unmounted.  After  examination  with  the  oil- 
immersion,  the  oil  may  be  removed  from  the  film  with  xylol.  Coles 
mounts  these  preparations  in  parolein. 

The  best  stains  are  Griibler's,  which  can  be  obtained  from  many 
agents  in  this  country.  Messrs.  Burroughs,  Wellcome  and  Co.  supply 
most  of  the  anilin  dyes  and  some  other  reagents,  iodine,  etc.,  in 
"  soloids,"  which  are  very  convenient  and  good. 

Gram's  method. — This  is  a  most  useful  method,  especially 
for  sections,  specimens  of  blood,  or  films  or  impression 
preparations,  as  the  tissue  or  ground  substance  can  be 
counter- stained  so  that  the  organisms  show  up  in  marked 
contrast.  Ordinary  films  of  cultures  do  not  usually  require 
this  method,  unless  debris  or  ground  substance  is  present  and 


GRAM'S  METHOD  103 

the  best  result  is  desired.  Unfortunately  Gram's  method 
is  not  applicable  for  all  organisms,  as  many  do  not  retain 
their  colour  by  the  process.  This  disadvantage,  however, 
is  counter- balanced  by  the  fact  that  it  forms  a  valuable 
means  of  distinguishing  organisms,  and  is  always  one  of 
the  points  to  be  noted  in  bacteriological  diagnosis.  Most 
of  the  moulds,  yeast,  streptothrix  and  sarcina  forms,  and 
cocci  stain  by  it,  though  there  are  exceptions  ;  the  spirilla 
and  protozoa  do  not  stain  by  it,  but  as  regards  the  bacilli 
no  rule  can  be  laid  down  (see  p.  105).  Films  are  stained 
for  five  to  ten  minutes,  and  sections  for  ten  minutes  to 
half  an  hour,  in  anilin-  or  carbol- gentian  violet  solution. 
The  superfluous  stain  is  then  drained  or  blotted  off,  not 
washed  away,  the  specimen  is  rinsed  with  Gram's  iodine 
solution  and  is  treated  with  fresh  iodine  solution  for  from 
one-half  to  two  minutes. 

GRAM'S  IODINE  SOLUTION 

Iodine                      ....  1  part 

Potassium  iodide    .....  2  parts 

Distilled  water 300  parts 

The  purple  colour  of  the  gentian  violet  changes  to  a 
dirty  yellowish- brown,  and  sections  become  much  like 
a  used  tea-leaf.  The  specimens  must  not  be  passed  on 
to  the  next  solution  until  they  have  assumed  the  brown 
colour.  Cover- glass  specimens  are  best  immersed  in  the 
solution  in  a  watch- glass,  film  side  up. 

The  specimens  are  removed  from  the  iodine  solution, 
drained,  and  then  immersed  in  alcohol,  preferably  methyl- 
ated spirit.  In  this  the  purple  colour  of  the  gentian 
violet  returns  and  is  dissolved  out,  so  that  they  ultimately 
become  colourless ;  this  is  aided  by  moving  them  gently 
about,  and  for  sections  two  or  more  baths  of  alcohol  may 
be  an  advantage,  a  fresh  one  being  substituted  when  the 
first  has  become  deeply  coloured.  Films  decolorise  much 
more  readily  than  sections,  and  they  should  be  removed 


104  A  MANUAL  OF  BACTERIOLOGY 

from  the  alcohol  when  no  more  colour  dissolves  out,  or 
the  stain  may  be  entirely  removed ;  usually  twenty  to 
forty  seconds  in  the  alcohol  suffices,  thick  preparations 
taking  longer  than  thin  ones.  After  decolorising,  films 
are  washed  in  water,  dried,  and  mounted,  or,  after  washing, 
the  ground  substance  may  be  counter-stained,  if  required, 
with  eosin  for  a  few  seconds,  or  Bismarck  brown  for  two 
or  three  minutes,  washed  again  in  water,  dried,  and 
mounted.  With  films  it  is  important  to  remember  on 
which  side  of  the  glass  the  film  is,  for  it  may  be  very 
difficult  to  ascertain  this  after  decolorisation.  Sections 
after  decolorising  are  passed  through  absolute  alcohol  and 
xylol  before  mounting,  or,  if  required  to  be  counter- 
stained,  are  immersed  in  eosin  for  fifteen  to  thirty  seconds, 
or  Bismarck  brown  for  three  to  five  minutes,  and  then 
passed  through  alcohol,  absolute  alcohol,  and  xylol. 

Sections  frequently  are  somewhat  difficult  to  decolorise 
with  alcohol  alone,  in  which  case  it  is  well  to  treat  them 
with  a  slightly  acid  alcohol  (3  per  cent,  of  hydrochloric 
acid)  for  a  few  seconds,  and  then  return  to  the  alcohol 
(Giinther's  method). 

The  iodine  in  Gram's  method  seems  to  act  as  a  mordant, 
precipitating  the  stain  in  a  relatively  insoluble  form  in 
certain  species  of  bacteria.  The  staining  of  organisms 
by  Gram  is  relative ;  some  forms  do  not  stain  at  all,  are 
Gram-negative — i.e.  the  colour  is  removed  by  the  alcohol 
with  the  greatest  facility ;  others  stain  intensely,  are 
Gram-positive,  but  even  these  may  become  decolorised 
by  prolonged  treatment  with  alcohol.  In  order  to  ascer- 
tain whether  an  organism  is  or  is  not  stained  by  Gram's 
method,  it  is  sometimes  useful  to  mix  with  it  in  making 
the  preparation  some  undoubted  Gram-staining  organism 
— e.g.  if  a  bacillus,  the  Micrococcus  pyogenes  ;  if  a  coccus, 
B.  anthracis  or  B.  subtilis.  The  admixed  organism  then 
serves  as  an  index. 


WEIGERT'S  METHOD  105 

The  following  organisms  are  Gram-positive :  B.  anthracis, 
B.  diphtherice,  B.  tetani,  B.  \Vefchii,  B.  botulinus,  B.  tuberculosis, 
B.  smegmatis,  B.  leprce,  B.  murisepticus,  Actinomyces,  B.  subtilis, 
B.  mesentericus,  B.  megaterium,  B.  mycoides,  the  pyogenic  cocci, 
the  streptococci,  including  the  pneumococcus,  most  cocci,  yeasts, 
moulds,  and  streptothrices. 

The  following  organisms  are  Gram-negative :  B.  typhosus, 
B.  enteritidis,  B.  dysenterice,  B.  coli,  B.  pestis,  B.  inftuenzce,  B.  mallei, 
B.  pseudo-tuberculosis,  B.  pyocyaneus,  B.  osdematis  maligni,  B. 
Chauvcei  (usually),  B.  prodigiosus,  B.  proteus,  the  septicaemic  bacilli, 
such  as  chicken  cholera,  the  spirilla  and  vibrios,  spirochaetes  and 
protozoa,  M.  gonorrhoeas,  M.  meningitidis,  M.  melitensis,  and 
M.  catarrhalis. 

Gram's  method  of  staining  depends  upon  the  formation  of  an 
iodine-pararosanilin-protein  compound  which  is  not  readily  dis- 
sociable in  the  case  of  the  Gram-positive  organisms.  Pararosanilin 
dyes,  such  as  gentian  violet,  methyl  violet  and  victoria  and 
thionine  blues,  are  alone  suitable  for  the  method. 

In  Claudius's  modification  of  Gram's  method,1  staining 
is  done  in  a  1  per  cent,  aqueous  solution  of  methyl  violet 
(films  for  one  minute,  sections  for  two  minutes).  The 
preparations  are  washed,  treated  with  a  half -saturated 
aqueous  solution  of  picric  acid  for  one  to  two  minutes, 
washed  again,  and  dried  with  filter- paper.  Decolorisation 
is  then  carried  out  in  the  case  of  films  with  chloroform, 
in  that  of  sections  with  clove  oil.  After  decolorising,  the 
preparations  are  treated  with  xylol  and  mounted.  By 
this  method  the  ordinary  Gram-positive  organisms  are 
stained  ;  also  the  bacilli  of  malignant  cedema  and  of  black 
quarter.  Counter-staining  may  be  carried  out  with  lithium 
carmine. 

W eigert's  modification  of  Gram's  method. — In  this  process 
the  sections,  whether  frozen  or  paraffin  ones,  should  be 
manipulated  on  the  slide.  They  are  stained  with  the 
anilin  gentian  violet  and  treated  with  Weigert's  iodine 
solution  (iodine  4-5  per  cent.,  potassium  iodide  6  per 
cent.)  as  in  the  simple  Gram's  method.  The  iodine  is  then 
1  Ann.  de  VInst.  Pasteur,  xi,  1897,  p.  332. 


106  A  MANUAL  OF  BACTERIOLOGY 

removed  with  filter-paper  and  the  sections  are  flooded 
with  anilin  oil  two  or  three  times.  This  removes  the 
colour  and  dehydrates.  The  anilin  oil  is  removed  by 
flooding  two  or  three  times  with  xylol. 

Thionine  blue  may  be  used  for  Gram's  method,  the 
carbol  solution  being  employed  (No.  5,  p.  100).  Sections 
are  stained  for  two  or  three  minutes,  then  treated  with 
an  iodine  solution  somewhat  stronger  than  Gram's  (200 
parts  of  water  instead  of  300  parts).  The  sections,  after 
remaining  in  this  for  one  to  two  minutes,  are  decolorised  in 
alcohol  containing  1  per  cent,  of  acetone  (methylated  spirit 
does  very  well),  and  subsequently  treated  as  in  Gram's 
method. 

The  Staining  of  Film  Specimens 

To  stain  films,  smear,  and  impression  preparations, 
the  film  is  flooded  after  fixing  with  a  drop  or  two  of  the 
solution,  or  the  preparation,  if  a  cover- glass,  may  be 
floated,  film  side  down,  on  the  solution  contained  in  a 
watch-glass ;  if  it  should  sink  it  makes  little  difference. 
Various  baths  or  pots  can  be  obtained  for  staining  slides. 
Warming  intensifies  the  staining  properties  of  all  staining 
solutions,  and  may  be  necessary  if  deep  staining  is  required 
or  if  the  temperature  of  the  laboratory  be  low  (see  also 
p.  110).  When  stained  sufficiently,  the  preparation  is 
rinsed  in  a  beaker  or  tumbler  of  water,  or  in  a  fine  stream 
of  water,  preferably  distilled,  to  remove  the  superfluous 
colour,  after  which  it  is  dried  and  mounted  in  a  drop 
of  solution  of  Canada  balsam  in  xylol.  The  preparation 
may  be  dried  either  by  gentle  warming  over  the  Bunsen 
flame  after  the  film  has  been  blotted  with  filter-paper,  or 
the  film  may  be  allowed  to  dry  spontaneously  in  the  air, 
in  which  case  it  should  always  be  set  up  on  edge  to  drain. 
The  preparations  must  be  completely  dried  before  being 
mounted  in  balsam. 


FILM  STAINING  107 

To  prevent  the  stain  flowing  all  over  a  slide,  two  lines 
may  be  drawn  across  the  slide  with  a  grease  pencil,  one 
on  either  side  of  the  area  to  be  stained. 

If  there  be  much  debris  or  other  material  which,  when 
stained,  would  interfere  with  a  clear  view  of  the  organisms, 
various  expedients  may  be  adopted.  One  is  to  stain  for 
a  short  time  with  a  solution  which  does  not  give  a  very 
dense  colour,  the  best  for  this  purpose  being  Loffler's 
methylene  blue,  or  Gram's  method  may  be  made  use  of 
if  the  organism  stains  by  it,  and  will  give  the  best  result 
of  any.  Another  plan  is  to  treat  the  specimen  with  acetic 
acid  before  staining  ;  it  may  be  just  dipped  in  glacial 
acetic  acid  and  immediately  washed  in  distilled  water,  or 
immersed  in  20  per  cent,  acetic  acid  for  five  to  ten  minutes, 
washed  in  distilled  water,  and  then  stained.  A  third  is, 
after  staining  and  washing,  to  rinse  the  preparation  in 
dilute  alcohol  (alcohol  1  part,  water  1  or  2  parts),  and 
immediately  to  wash  again  in  water  to  stop  the  further 
action  of  the  alcohol.  If  the  film  be  thick,  two  or  three 
rinses  in  the  dilute  alcohol  may  be  necessary.  This  process 
gives  excellent  results  with  the  sarcinae,  but  the  staining 
agent  should  be  anilin  gentian  violet  or  dilute  carbol- 
fuchsin  and  not  LofHer's  blue,  unless  it  is  allowed  to  act 
for  fifteen  to  twenty  minutes.  The  treatment  with  acetic 
acid  before  staining  may  be  combined  with  decolorisation 
with  alcohol  after. 

Preparations  can  always  be  examined  in  water  with  the 
J-in.  objective,  after  washing  and  before  permanently 
mounting,  in  order  to  see  whether  they  are  satisfactory. 
If  the  film  is  on  a  slide,  a  drop  of  water  is  put  on  and 
covered  with  a  cover-glass,  if  on  a  cover-glass,  this  is 
mounted  in  a  drop  of  water  on  a  slide.  If  satisfactory, 
the  preparation  can  be  dried,  and  mounted  in  balsam ;  or 
if  not  stained  sufficiently,  or  if  stained  too  deeply,  it  can  be 
stained  again,  or  further  decolorised,  as  the  case  may  be. 


108  A  MANUAL  OF  BACTERIOLOGY 

Treatment  of  Sections  for  Staining  and 
Mounting 

(a)  Frozen  sections. — If  preserved  in  spirit  they  should 
be  rinsed  in  distilled  water  or  in  fresh  alcohol  before 
staining,  according  as  the  staining  solution  is  an  aqueous 
or  an  alcoholic  one.  After  staining  they  are  well  rinsed 
in  water  or  alcohol  to  remove  the  excess  of  stain,  and  are 
then  dehydrated  and  cleared  before  being  mounted.  For 
dehydrating,  if  they  have  been  washed  in  water,  they 
should  be  well  rinsed  in  methylated  spirit l  to  remove  the 
excess  of  water,  and  then  transferred  to  absolute  alcohol 
for  a  few  seconds  to  two  minutes,  the  time  varying  with 
the  size  and  thickness  of  the  section.  In  many  cases — for 
instance,  when  the  anilin  dyes  have  been  used  for  staining 
• — the  sections  must  be  passed  as  rapidly  as  possible,  con- 
sistent with  thorough  dehydration,  through  the  absolute 
alcohol  to  avoid  removing  too  much  of  the  colour.  If 
it  is  important  to  avoid  any  decolorisation,  anilin  oil  may 
be  used  for  dehydration,  as  in  Weigert's  method  (pp.  105 
and  106).  For  clearing,  xylol  or  cedar  oil  is  the  best  agent, 
for  neither  dissolves  the  anilin  dyes  ;  they  will  only  clear, 
however,  out  of  absolute  alcohol :  hence  the  preliminary 
rinsing  of  water- washed  sections  with  methylated  spirit 
to  prevent  dilution  of  the  subsequent  bath  of  absolute 
alcohol.  Oil  of  cloves  can  also  be  employed,  but  has  the 
disadvantage  that  it  dissolves  the  anilin  dyes,  and  the 
colour  of  stained  sections  treated  with  it  is  apt  to  be  less 
permanent ;  it  has  the  advantage,  however,  of  clearing 
out  of  methylated  spirit,  absolute  alcohol  being  unnecessary. 
The  alcohol  and  clearing  agents  are  conveniently  placed 
in  watch-glasses  or  small  shallow  glass  capsules.  The 

1  Absolute  alcohol  may  of  course  be  employed  instead  of  the  first 
bath  of  methylated  (or  rectified)  spirit,  but  methylated  answers  just 
as  well  and  is  less  expensive  (but  see  note,  p.  86). 


PARAFFIN  SECTIONS  109 

section  is  known  to  be  cleared  when  it  appears  quite 
transparent  and  almost  invisible  when  the  watch-glass 
or  capsule  containing  it  is  held  over  a  dark  surface.  If 
after  two  minutes  in  the  clearing  agent  the  section  still 
appears  cloudy  and  opaque,  it  has  not  been  sufficiently 
dehydrated,  and  should  be  returned  to  a  fresh  bath  of 
absolute  alcohol  for  a  short  time,  and  then  transferred 
again  to  the  clearing  agent.  Care  should  be  taken  that 
watch-glasses,  etc.,  used  for  the  absolute  alcohol  and 
clearing  agent  are  perfectly  dry.  The  clearing  agent, 
especially  clove  oil,  can  be  used  many  times  before 
becoming  useless. 

For  transferring  the  sections  from  one  solution  to 
another  an  ordinary  needle,  fixed  in  a  light  wooden  handle, 
suffices,  or,  better  still,  a  piece  of  glass  drawn  out  at  one 
end,  the  section  being  carefully  lifted  by  one  corner  to 
prevent  crumpling ;  but  for  the  final  process  of  mounting 
it  is  necessary  to  use  a  section  lifter  or  cigarette-paper. 
The  section,  spread  out  with  care,  is  raised  by  means  of 
the  section  lifter  or  cigarette-paper  introduced  under  it, 
and  transferred  to  the  slide,  any  crinkles  are  removed 
by  spreading  with  a  needle,  the  superfluous  clearing  agent 
is  drained  off,  a  drop  of  xylol  balsam  put  on,  and  it  is  then 
covered  with  a  clean  cover-glass.  If  clove  oil  has  been  used 
as  the  clearing  agent,  the  section,  after  draining,  should 
be  blotted  with  two  or  three  thicknesses  of  filter-paper 
to  remove  as  much  oil  as  possible  before  putting  on  the 
balsam.  In  blotting  firm  pressure  should  be  used,  and 
the  section  will  then  adhere  to  the  glass  slide  and  not  to 
the  blotting-paper.  With  delicate  sections  all  the  pro- 
cesses of  staining,  dehydrating,  clearing,  etc.,  may  be 
carried  out  on  the  slide. 

(b)  Paraffin  sections. — The  section  fixed  on  the  slide 
(p.  93)  must  be  freed  from  paraffin  before  staining  and 
mounting.  The  slides  with  attached  sections  are  treated 


110  A  MANUAL  OF  BACTERIOLOGY 

as  follows  :  Immerse  in  (1)  xylol  for  one  or  two  minutes, 
drain  ;  (2)  absolute  alcohol  one  to  two  minutes  to  remove 
the  xylol,  drain ;  (3)  distilled  water.  They  are  now 
ready  for  staining,  and  are  to  be  flooded  with  the  staining 
solution  or  immersed  in  it,  and  after  staining  they  are 
treated  in  the  same  manner,  but  in  the  reverse  order,  viz. 
(1)  distilled  water;  (2)  methylated  spirit;  (3)  absolute 
alcohol ;  (4)  xylol.  On  being  removed 
from  the  xylol  the  slide  is  drained  for  a 
few  seconds,  a  drop  of  xylol  balsam  is  then 
put  on,  and  the  section  covered  with  a 
clean  cover-glass.  Glass  pots  (Fig.  20} 
filled  with  the  alcohol,  xylol,  etc.,  are 
convenient  for  the  treatment  of  paraffin 
sections,  the  slide  with  the  section  upon  it 
being  immersed  in  the  fluid. 

Section  Staining 

When  Gram's  method   is  applicable   it 
FIG.  20.— Glass  pot  ,  ,       ., 

for  clearing,  etc.     gives  by  far  the  best   results,  and  should 

always  be  employed.  If,  however,  the 
organisms  are  decolorised  in  Gram's  process  some  other 
method  must  be  adopted.  One  of  the  best  is  to 
stain  for  from  ten  minutes  to  six  or  eight  hours  in 
Lomer's  methylene  blue.  Fresh  easily  staining  organisms 
will  be  sufficiently  stained  in  ten  or  fifteen  minutes,  but 
when  the  organism  is  difficult  to  stain,  as  glanders,  six  to 
eight  hours  may  not  be  too  long  a  time.  Warming  intensi- 
fies the  staining  properties  of  all  staining  solutions ;  for 
frozen  sections  the  watch-glass  of  stain  may  be  warmed 
on  a  sand-bath  or  asbestos  cardboard,  or  in  the  blood-heat 
incubator.  Sections  on  the  slide  may  be  flooded  with  the 
stain  and  warmed  on  a  piece  of  asbestos  cardboard  placed 
over  a  Bunsen  flame,  or  a  penny  may  be  heated  in  the 


SECTION  STAINING  111 

Bunsen  and  the  preparation  laid  on  it,  the  coin  being 
re-heated  as  often  as  required.  The  stain  may  be  pre- 
vented from  flooding  the  slide  by  confining  it  between 
grease-pencil  lines  as  described  for  films  (p.  107).  After 
staining,  the  sections  are  well  rinsed  in  distilled  water 
and  then  slightly  decolorised  by  rinsing  for  half  a  minute 
or  so  in  a  watch-glass  of  1  per  cent,  acetic  acid  in  distilled 
water.  They  are  then  again  washed  and  passed  as  rapidly 
as  possible  through  alcohol,  cleared  in  xylol,  and  mounted. 
Carbol-methylene  blue  or  carbol-thionine  blue  may  be 
used  instead  of  the  Lofner's  solution,  the  staining  taking 
from  a  few  minutes  to  half  an  hour.  If  a  contrast  stain 
be  desired  the  sections  may  be  treated  for  a  few  seconds 
with  the  eosin  solution  after  the  dilute  acetic.  If  staining 
be  prolonged  evaporation  must  be  prevented.  In  the 
case  of  a  section  mounted  on  the  slide  and  flooded  with 
stain,  the  slide  should  be  placed  on  a  piece  of  wet 
blotting-paper  on  a  tile  and  covered  with  the  lid  of  a 
Petri  dish. 

The  micro-organisms  in  sections  stained  with  Loffler's 
blue  are  very  liable  to  become  decolorised  unless  the 
dehydration  is  expeditiously  performed.  To  avoid  this 
Unna's  method  may  be  adopted.  After  staining  and 
decolorising  with  acidulated  water  as  described,  the 
sections  are  placed  on  the  slide  (if  not  already  mounted 
thereon),  gently  warmed,  and  so  dried  ;  they  are  then 
treated  with  xylol  and  mounted  in  balsam.  The  tissue 
elements,  however,  are  apt  to  suffer. 

A  better  method  is,  after  decolorising  with  the  dilute 
acid,  to  dehydrate  with  anilin  instead  of  with  alcohol, 
the  section  being  treated  with  fresh  anilin  two  or  three 
times,  then  with  a  mixture  of  equal  parts  of  anilin  and 
xylol,  and  finally  with  two  or  three  baths  of  xylol. 


112  A  MANUAL  OF  BACTERIOLOGY 

Capsule  Staining 

Many  organisms,  especially  in  the  tissues  or  body  fluids, 
are  invested  with  a  capsule  of  gelatinous  matter,  probably 
derived  from  the  membrane  of  the  bacterial  cell,  and 
differing  in  composition  in  different  species.  The  capsule 
may  be  as  thick  as  the  bacterial  cell  itself,  and  appears, 
in  the  unstained  state  or  after  staining  by  the  ordinary 
methods,  as  a  clear  halo  or  zone  surrounding  the  organism. 
Organisms  in  films  of  albuminous  matter  often  appear  to 
be  surrounded  by  a  clear  halo,  which  must  not  be  mistaken 
for  a  capsule.  As  organisms  frequently  lose  their  capsules 
on  ordinary  culture  media,  Moore  recommends  cultivating 
in  fluid  serum  to  obtain  the  re- development  of  the  capsule. 
In  order  to  stain  the  capsule  one  of  the  following  methods 
may  be  adopted. 

1.  Stain  the  preparations  by  just  dipping  in  the  following  solution : 

Carbol-fuchsin  .  1  part 

Distilled  water  .  .  1  part 

Rinse  in  water  and  then  stain  for  fifteen  seconds  in  a  very  weak 

aqueous  solution  of  gentian  violet  (0-1  per  cent.).     Rinse  in  water, 

dry,  and  mount. 

2.  McConTcey's  method. — The  following  solution  is  prepared  : 

Methyl  green  .          .  .1-5  grm. 

Dahlia  ....  .0-5  grm. 

Distilled  water        .          .  .     100  c.c. 

When  dissolved,  10  c.c.  of  a  saturated  alcoholic  solution  of  fuchsin 
are  added,  and  the  whole  is  made  up  to  200  c.c.  with  distilled 
water.  The  stain  should  not  be  used  for  a  fortnight,  and  should 
be  kept  in  a  dark  place.  Specimens  are  stained  for  five  minutes 
or  longer,  then  thoroughly  washed  in  a  stream  of  water,  dried,  and 
mounted. 

3.  Friedlander's  method  (for  tissues).— Mix, 

Concentrated  alcoholic  solution  of  gentian 

violet          .  50  parts 

Distilled  water        .  100  parts 

Acetic  acid    .  .10  parts 

Stain  the  sections  in  this  solution  in  the  warm  incubator  for 


SPORE  STAINING  113 

twenty-four  hours.     Rinse  well  in  1   per  cent,   acetic  acid,  pass 
through  alcohol  and  xylol,  and  mount  in  balsam. 


Spore  Staining 

When  spore- bearing  bacteria  are  stained  by  the  ordinary 
methods  the  spores  are  just  tinted,  or  remain  uncoloured 
with  the  outlines  more  or  less  stained.  This  seems  to  be 
due  to  the  fact  that  the  spores  are  surrounded  with  a 
slightly  permeable  membrane  which  inhibits  the  entrance 
of  the  staining  agent.  By  staining  by  some  method  which 
causes  the  penetration  of  the  stain,  and  then  cautiously 
decolorising,  it  is  possible  to  remove  the  colour  from  every- 
thing except  the  spores,  the  impermeable  membrane  of 
which  in  the  same  way  prevents  the  full  action  of  the 
decolorising  agent. 

(a)  Simple  method. — A  film  is  prepared  in  the  ordinary  way.  If 
a  cover-glass,  it  is  floated  on  a  watch-glass,  or,  if  a  slide,  it  is  flooded 
with  carbol-fuchsin,  and  the  stain  is  warmed  for  twenty  minutes. 
After  being  washed  in  water  the  preparation  is  rinsed  for  a  second 
or  two  in  1  per  cent,  sulphuric  acid  and  again  washed  at  once  in 
water.  If  there  is  still  a  good  deal  of  the  red  colour  remaining, 
the  film  may  be  once  more  rinsed  in  the  acid,  but  if  nearly  colour- 
less it  should  be  mounted  in  water  and  examined  with  the  £-in. 
objective.  If  the  spores  alone  are  well  stained  the  preparation 
may  be  counter-stained  with  Loffler's  methylene  blue  for  two  to 
five  minutes,  washed,  dried,  and  mounted.  If,  however,  the 
bacilli  as  well  as  the  spores  retain  the  red  colour,  the  preparation 
must  be  further  decolorised  in  the  acid,  while  if  everything  has 
been  decolorised,  it  may  be  re-stained  with  warm  carbol-fuchsin. 

The  spores  sometimes  stain  better  if  the  preparation  be  fixed 
by  passing  through  the  flame  twelve  times  instead  of  three,  as  is 
usual.  To  obtain  good  preparations  and  ones  showing  the  spores 
in  situ,  the  specimens  should  be  made  as  soon  as  spores  have 
definitely  developed  in  the  cultures. 

Spore  staining  often  requires  a  good  deal  of  patience,  and  in 
many  instances  it  is  difficult  to  obtain  a  satisfactory  preparation 
by  this  simple  method,  in  which  case  that  of  MoeJler  should  be 
made  use  of,  and  rarely  fails. 

8 


114  A  MANUAL  OF  BACTERIOLOGY 

(b)  Moeller's  method. — Prepare  the  cover-glass  or  slide  specimen 
in  the  ordinary  way.  Treat  with  absolute  alcohol  for  two  minutes, 
and  then  with  chloroform  for  two  minutes.  Wash  in  water  and 
treat  with  a  5  per  cent,  solution  of  chromic  acid  for  two  minutes, 
wash,  and  then  stain  with  warm  carbol-fuchsin  for  ten  minutes. 
Wash,  decolorise  carefully  in  1  per  cent,  sulphuric  acid,  again  wash 
and  counter-stain  with  Loffler's  methylene  blue  for  one  minute  ; 
wash,  dry,  and  mount.  Some  organisms,  such  as  the  B.  mesen- 
tericus,  stain  better  if  treated  with  the  chromic  acid  for  five  to  ten 
minutes. 

Flagella  Staining 

Many  organisms  possess  delicate  protoplasmic  processes 
— flagella — in  greater  or  less  number ;  but  these  are  not 
visible  when  the  organism  is  examined  in  the  living  con- 
dition (except  by  the  use  of  dark-ground  illumination), 
nor  when  the  ordinary  staining  methods  are  employed. 
In  order  to  demonstrate  them  it  is  necessary  to  make  use 
of  some  special  method,  in  which  a  mordant  is  essential. 
One  of  the  earliest  devised  was  that  of  Loffler,  which  with 
care  gave  fair  results.  It  is  not,  however,  nearly  so 
satisfactory  as  some  more  recent  ones,  so  is  omitted. 

For  all  methods  of  flagella  staining  the  cover- glasses  or 
slides  must  be  absolutely  clean,  the  cultures  recent,  and 
the  growth  sufficiently  diluted  to  obtain  the  organisms  in 
an  isolated  condition. 

(a)  Stephens's  method. — This  is  a  modification  of  the  well-known 
Van  Ermengem  method,1  and  has  been  communicated  to  the  writer 
by  Dr.  J.  W.  W.  Stephens. 

To  clean  slides. — Rub  the  slides  with  a  clean  cloth  and  place  on 
a  piece  of  clean  wire  gauze  and  heat  with  a  smokeless  flame  for 
some  minutes  (by  this  means  grease  is  completely  removed). 
Remove  the  slides  when  cool,  not  before. 

To  make  the  suspension. — All  methods  are  unsatisfactory.  Rub  a 
little  of  the  culture  in  a  small  drop  of  tap-water  in  a  watch-glass. 
Then  transfer  a  drop  with  the  smallest  possible  platinum  loop  to 
a  minute  drop  of  water  on  the  slide.  Mix  and  spread  with  the 

1  Cenlr.  f.  Bakt.,  xv,  1894,  p.  969. 


FLAGELLA  STAINING  115 

platinum  wire  as  quickly  as  possible.     The  film  thus  made  should 
dry  immediately  if  a  small  drop  only  of  water  has  been  used. 

Age  of  the  culture. — A  twenty -four  hours'  culture  does  quite  well 
(a  younger  one  is  perhaps  better,  but  flagella  can  be  shown  for  a 
week  or  fortnight  or  more). 
I.  The  mordant : 

Osmic  acid,  2  per  cent.          .          .          .1  part 
Tannic  acid,  20  per  cent,  watery  solution     3  or  4  parts 
II.  Silver  solution  :  Silver  nitrate  .          .     1  per  cent. 

III.  Gallic  acid,  2  per  cent,  solution         .          .     1  part 
Ammonia  fort.    ...  .1  part 

To  be  mixed  before  using  and  to  be  used  immediately. 

To  stain. — Place  the  mordant  on  the  film  for  one  or  two  minutes 
or  less  (time  unimportant). 

1.  Wash  in  tap- water  thoroughly. 

2.  Shake  off  as  much  water  as  possible. 

3.  Place  a  few  drops  of  silver  nitrate  on  the  slide  for  a  few  seconds 
or  longer. 

4.  Shake  off  all  excess. 

5.  Allow  one  drop  of  the  ammonia-gallic  solution  to  fall  on  the 
middle  of  the  slide  from  a  small  pipette.     A  wave  spreads  away 
from  the  centre  to  each  end  of  the  slide.     As  soon  as  the  film  is 
seen  standing  out  clearly  and  black  in  the  centre  (in  a  few  seconds), 
wash  off  in  tap-water. 

6.  Add  again  a  drop  or  two  of  the  silver  solution  and  allow  it  to 
act  for  half  a  minute  or  thereabouts. 

7.  Wash  in  tap-water,  blot,  and  dry  over  the  flame. 

8.  It  is  best  not  to  mount  in  balsam  or  in  cedar-wood  oil,  as  the 
preparations  rapidly  fade  in  these. 

If  done  with  any  care,  the  film  should  now  appear  black  and 
distinct  to  the  naked  eye  with  no  precipitate,  and  the  flagella  will 
be  found  to  be  stained  distinctly  and  intensely  with  hardly  any 
ground  substance,  or  at  least  insufficient  to  interfere  with  a  clear 
view  of  them. 

(6)  Pitfield's  method. — Two  solutions  are  freshly  prepared  : 

A.  Saturated  aqueous  solution  of  alum          .          .     10  c.c. 
Saturated  alcoholic  solution  of  gentian  violet  .       1  c.c. 

B.  Tannic  acid  .......       1  grm. 

Distilled  water       .          .          .          .          .          .10  c.c. 

The  solutions  should  be  made  with  cold  water,  filtered,  and 


116  A  MANUAL  OF  BACTERIOLOGY 

preserved  in  separate  bottles.  For  use  equal  quantities  are  mixed 
together.  The  specimens  are  flooded  with  the  mixture  and  held 
over  the  flame  until  it  nearly  boils  ;  they  are  then  laid  aside,  with 
the  hot  stain  on  them,  for  one  minute,  and  are  finally  washed  in 
water.  After  washing,  the  preparations  are  flooded  with  anilin 
gentian  violet  for  one  second,  washed  in  water,  dried  and  mounted, 
(c)  McCrories  method  x  (modified  by  Morton  2). — Prepare  the 
following  solutions : 

A.  Tannic  acid  .......       1  grm. 

Potash  alum          ......       1  grm. 

Distilled  water       .  .     40  c.c. 

B.  "  Night  "  blue       .  ...     0-5  grm. 
Absolute  alcohol    .          .          .          .          .          .20  c.c. 

Mix  and  filter. 

The  prepared  slides  are  stained  with  this  solution  (which  should 
always  be  filtered  before  use)  for  two  minutes,  the  solution  being 
changed  two  or  three  times,  washed  gently  in  running  water,  and 
then  counter-stained  in  anilin  gentian  violet  for  one  to  two  minutes, 
washed,  dried,  and  mounted. 


Preservation  of  Cultures 

Gelatin  and  agar  cultures  may  be  satisfactorily  preserved  by 
submitting  them  to  the  action  of  formaldehyde  vapour  for  some 
hours  by  soaking  the  wool  plug  of  the  culture  tube  in  formalin  and 
plugging  the  tube  with  it.  The  tube  may  then  be  sealed  with 
gutta-percha  tissue,  sealing-wax,  or  paraffin  wax,  or  best  of  all  in 
the  blowpipe  flame.  Plate  cultivations  may  also  be  exposed  to  the 
vapour  and  the  lid  of  the  dish  afterwards  cemented  on,  or  the 
cultures  may  be  made  in  the  flat  bottles  ("Soyka's  bottles") 
devised  for  the  purpose,  and  after  development  treated  like  tube 
cultures. 


Preservation  of  Pathological  Specimens 

These  may  be  preserved  in  the  ordinary  way  in  spirit,  but  a  much 
better  method,  by  which  the  natural  colour  of  the  specimen  is 
retained,  is  the  following.  The  specimens  are  first  washed  in  water, 

1  Brit.  Med.  Journ.,  1897,  vol.  i,  p.  971. 

2  Trans.  Jenner  Inst.  Prev.  Med.,  vol.  ii,  p.  242. 


PRESERVATION  OF  SPECIMENS  117 

and  then  placed  in  the  following  solution  for  twenty-four  to  forty- 
eight  hours  : 

Formalin       ......         6  parts 

Sodium  chloride      .....         1  part 

Sodium  sulphate     .....         2  parts 

Magnesium  sulphate        ....         2  parts 

Tap- water      ....  .100  parts 

After  being  taken  from  the  formalin  solution  the  specimens  are 
placed  in  methylated  spirit  for  ten  minutes,  and  then  in  a  fresh 
bath  of  methylated  ;  in  this  the  colour  to  a  large  extent  returns, 
and  they  should  be  carefully  watched  and  not  allowed  to  remain 
in  it  for  more  than  an  hour.  They  are  then  mounted  in  the 
following  mixture : 

Glycerine       ......       400  c.c. 

Potassium  acetate  .....       200  grm. 

Water 2000  c.c. 

A  trace  of  formalin  should  be  added  to  this. 

The  writer  has  preserved  meat  infected  with  B.  prodigiosus  very 
satisfactorily  by  the  following  method.  Slices  were  cut  off  and 
placed  in  the  formalin  solution  given  above  for  a  few  hours.  They 
were  then  well  drained  and  placed  in  suitable  glass  capsules. 
Ordinary  nutrient  gelatin  was  melted  and  sufficient  poured  in  to 
cover  the  specimens,  and  when  it  had  set  a  little  formalin  was 
poured  on  and  allowed  to  remain  for  a  few  days.  It  was  then 
poured  off  and  the  glass  top  cemented  down. 

For  further  information  on  preparation  of  tissues,  section  cutting, 
staining  methods,  etc.,  see  The  Microtomisfs  Vade-Mecum,  Bolles- 
Lee  ;  Practical  Histology,  Schafer  ;  Methods  of  Morbid  Histology 
and  Clinical  Pathology,  Walker  Hall  and  Herxheimer  ;  and  Lehrbuch 
der  Mikroskopischen  Technik,  Rawitz. 


CHAPTER  IV 

METHODS  OF  INVESTIGATING  MICROBIAL  DISEASES— 
THE  INOCULATION  AND  DISSECTION  OF  ANIMALS- 
HANGING -DROP  CULTIVATION  —  INTERLAMELLAR 
FILMS— THE  MICROSCOPE 

THE  systematic  study  of  a  condition  dependent  on  the 
activity  of  micro-organisms  is  in  many  instances  no  light 
matter.  When  only  one  or  two  forms  are  present  and 
these  are  readily  cultivated  it  may  be  comparatively 
easy,  but  when  there  are  many  the  investigation  may 
become  exceedingly  complicated.  The  first  step  to  be 
taken  is  to  ascertain  by  careful  microscopical  examina- 
tion the  general  characters  of  any  organisms  that  may 
be  present  in  the  material,  and  their  distribution  both 
in  the  fresh  condition  and  in  stained  preparations,  and 
if  possible  at  different  stages  of  the  disease.  In  disease 
conditions,  for  example,  the  blood  and  secretions  may  be 
examined  both  before  and  after  death,  but  in  the  latter 
it  must  be  remembered  that  soon  after  the  fatal  event 
adventitious  organisms  rapidly  make  their  appearance, 
gaining  access  from  the  air  and  from  the  intestinal  tract. 
If  organisms  be  detected  an  attempt  should  be  made  to 
determine  whether  there  is  any  predominant  form  and 
if  this  is  constantly  present  at  different  stages.  If 
organisms  are  found,  it  simplifies  matters,  but  if  not,  it 
cannot  therefore  be  said  that  they  are  absent,  for  they 
may  be  few  in  number,  and  consequently  be  missed  in  a 
microscopical  examination ;  or  they  may  be  confined  to 

118 


INVESTIGATION  OF  MICROBIAL  DISEASES    119 

a  particular  locality  or  tissue,  or  are  present  only  at  one 
stage  of  the  infection.  In  addition  to  the  microscopical 
examination,  cultures  must  be  made  on  various  media, 
those  media  being  chosen  which  will  probably  be  suitable 
for  the  growth  of  the  organism  present  in  the  particular 
condition ;  for  example,  in  the  examination  of  animal 
diseases,  media  rich  in  protein,  such  as  blood-serum, 
nutrient  agar  and  gelatin,  will  be  the  most  serviceable. 
In  the  examination  of  plant  diseases,  vegetable  infusions 
prepared  from  the  plant  itself  or  from  other  sources,  and 
enriched  by  the  addition  of  vegetable  proteins,  and  carbo- 
hydrates, should  be  chosen.  In  fermentations,  beer-wort, 
grape  or  fruit  juice,  and  saccharine  solutions  should  be 
made  use  of  ;  while  for  the  nitrifying  organisms,  solutions 
containing  nitrates  and  nitrites,  salts  of  ammonia,  urea, 
and  asparagin  will  have  to  be  employed.  In  addition, 
it  will  in  most  cases  be  advisable,  and  in  all  safer,  in  order 
to  isolate  the  various  species,  to  make  plate  cultivations, 
either  in  Petri  dishes  (p.  78),  or  by  streaking  several  sloped 
tubes  of  agar,  etc.  (p.  81).  Having  obtained  pure  cultiva- 
tions it  will  be  necessary  to  determine  the  species  of 
organism,1  if  it  has  been  previously  isolated  and  described, 
or  to  give  a  careful  description  of  it,  if  it  be  a  new  one, 
for  the  use  of  subsequent  investigators.  In  the  identifica- 
tion or  description  of  an  organism  all  the  following  features 
must  be  carefully  noted  : 

1.  The  morphology  of  the  organism  under  various  conditions,  its 
size,  form,  and  motility,  the  presence  of  flagella,  and  their  number, 
arrangement,  and  character. 

2.  The  presence  or  absence  of  spore  formation,  its  nature,  the 

1  The  descriptions  of  a  large  number  of  species  of  bacteria  have  been 
collected  and  tabulated  in  convenient  form  by  Chester  in  A  Manual 
of  Determinative  Bacteriology  (Macmillan  and  Co.,  1901).  The  terms  he 
suggests  for  describing  bacterial  growths,  etc.,  might  well  be  adopted 
by  bacteriologists.  A  committee  of  the  Society  of  American  Bacterio 
legists  has  drawn  up  an  elaborate  chart  for  the  description  of  species 
of  organisms. 


120  A  MANUAL  OF  BACTERIOLOGY 

conditions  under  which  it  occurs,   and  any  peculiarities  in  the 
germination  of  the  spores,  and  their  size  and  location  in  the  cell. 

3.  The  peculiarities  of  staining,  and  the  staining  reaction  with 
Gram's  and  the  Ziehl-Neelsen  methods. 

4.  The  characters  of  the  colonies  in  gelatin,  agar,  and  other 
media,  both  surface  and  deep. 

5.  The  characters  of  the  growth  on  a  variety  of  culture  media 
at  different  temperatures — for  example,  for  a  pathogenic  organism, 
on  blood-serum,  agar,  and  gelatin  (surface  and  stab  cultures),  in 
broth  and  on  potato  ;  liquefaction  or  not  of  the  gelatin  ;  the  growth 
in  milk,  with  or  without  curdling,  and  the  reaction  therein  ;   and 
the  fermentation  reactions  on  carbohydrates,  glucosides,  alcohols, 
etc.  ;    the  nature  of  the  gas,  if  any,  formed  therefrom,  and  the 
H:C02   ratio. 

6.  The  behaviour  towards  oxygen — is  it  aerobic  or  anaerobic  ? 

7.  The  range  of  growth  at  different  temperatures. 

8.  The  reducing  power  by  growing  in  litmus  broth  which  becomes 
decolorised,  or  by  the  formation  of  nitrites  in  a  solution  containing 
nitrates. 

9.  The  production  of  indole  with  or  without  nitrites. 

10.  The  production  of  pigment  and  the  conditions  under  which 
it  occurs. 

11.  The  pathogenic  action  on  various  animals  if  it  be  a  disease 
germ,  or  the  changes  which  it  produces  if  it  be  an  organism  connected 
with  other  conditions. 

12.  The  chemical  changes  which  it  induces. 

13.  The  thermal  death-point  and  the  action  of  germicides  and 
antiseptics  upon  it  (see  Chapter  XXII). 

For  descriptive  purposes,  "  standard "  culture  media 
should  always  be  employed,  and  the  acidity  or  alkalinity 
of  the  medium  stated  (p.  64). 

It  must  never  be  forgotten  that  under  cultivation  the 
properties  of  organisms  may  be  considerably  modified,  and 
due  allowance  must  be  made  for  this.  For  example, 
pathogenic  organisms  may  lose  their  virulence  more  or 
less  completely,  pigment  production  be  lost,  and  fer- 
mentive  action  modified  (see  also  p.  6). 

To  obviate  these  difficulties  the  organisms  should  be 
cultivated  under  as  nearly  natural  conditions  as  possible 
and  sub- cultivation  avoided  so  far  as  can  be.  No  general 


COLLODION  SACS  121 

rule  can  be  given  as  to  the  duration  of  life  of  cultures  on 
artificial  media.  Most  organisms  will  retain  their  vitality 
for  at  least  three  or  four  weeks  without  being  transferred 
to  a  fresh  soil,  some  for  many  months ;  a  few  must  be 
sub- cultured  every  week,  or  they  will  die  out ;  while 
there  are  still  a  small  number  which  have  so  far  rarely  or 
never  been  cultivated.  On  the  whole,  organisms  retain 
their  vitality  best  on  gelatin. 

For  an  organism  to  retain  its  virulence  it  is,  as  a  rule, 
necessary  to  pass  it  through  a  susceptible  animal  at  longer 
or  shorter  intervals,  and  to  enhance  the  virulence  recourse 
must  be  had  to  a  succession  of  passages  through  susceptible 
and  then  less  susceptible  animals.  In  this  way  the  viru- 
lence of  organisms  has  been  increased  to  a  point  far  greater 
than  is  ever  met  with  naturally,  as  in  the  case  of  the 
Streptococcus  pyogenes.  If  an  organism  retains  its  virulence 
even  slightly,  it  is  generally  possible,  by  employing  large 
doses,  to  enhance  this  by  passage  through  a  susceptible 
animal.  Another  method  may  also  be  adopted,  namely, 
to  inject  along  with  it  some  other  pathogenic  form,  such 
as  the  Streptococcus  pyogenes  ;  the  combination  will  kill 
the  animal,  and  the  slightly  virulent  organism  can  be 
recovered  and  will  be  found  to  have  increased  in  virulence. 
A  third  method  is  to  inject  the  organism  into  a  susceptible 
animal  together  with  a  lethal  dose  of  toxin  obtained  from 
a  virulent  form  of  the  same  species,  or  with  some  substance, 
such  as  lactic  acid,  which  lowers  the  vitality  of  the  tissues. 
The  slightly  virulent  organism  will  then  be  able  to  grow 
under  the  more  favourable  conditions,  and  a  form  which 
has  become  completely  non-virulent  can  be  made  to 
regain  its  lost  virulence. 

Collodion  sacks  are  now  frequently  used  to  study  the 
action  upon  animals  of  the  dialysable  products  produced 
by  micro-organisms  which  do  not  form  any  appreciable 
amount  of  toxin  in  vitro,  for  cultivating  species  which 


122  A  MANUAL  OF  BACTERIOLOGY 

are  difficult  to  grow  by  ordinary  methods,  for  studying 
the  phenomena  of  infection  when  the  micro-organisms  are 
protected  from  the  phagocytes,  and  for  other  purposes. 
A  glass  rod  or  small  test-tube,  according  to  the  size  desired, 
is  dipped  into  a  beaker  containing  the  ordinary  (not 
flexible)  collodion,  is  then  withdrawn  and  allowed  to  dry, 
and  the  process  is  repeated  two  or  three  times.  In  order 
to  detach  the  collodion  from  the  glass,  the  whole  is  dipped 
for  a  few  seconds  alternately  into  strong  spirit  and  into 
water,  the  collodion  loosens,  and  may  be  easily  peeled 
off  the  glass.  The  sack  may  be  sterilised  by  placing  in  a 
test-tube  and  heating  to  150°  C.  in  the  hot-air  steriliser. 

For  the  inoculation  of  animals  various  methods  may  be 
adopted.  Thus,  after  clipping  the  hair,  the  organism  may 
be  introduced  by  rubbing  into  the  skin  after  scarification, 
or,  a  small  incision  having  been  made  through  the  skin, 
a  small  quantity  of  a  culture  may  be  introduced  on  a 
platinum  needle  ;  or  a  broth  culture  or  an  emulsion,  made 
with  sterilised  water  or  broth,  may  be  injected  with  a 
sterilised  syringe  subcutaneously,  intra-peritoneally,  or 
into  the  muscular  or  other  tissues  or  organs  as  required, 
since  the  seat  of  inoculation  may  have  to  be  varied  for  the 
different  species  to  produce  their  pathogenic  effect.  For 
injection  purposes  a  syringe  like  an  antitoxin  syringe, 
i.e.  with  asbestos  or  metal  piston  and  glass  barrel  that 
can  be  boiled,  may  be  used.  Several  sizes,  1  c.c.,  2  c.c., 
and  5  c.c.  at  least,  are  required.  An  all-glass  syringe  is 
a  still  better  form,  but  is  expensive.  For  accurate  dosage, 
the  piston-rod  should  be  graduated  and  have  a  nut 
travelling  on  a  screw  up  and  down  it.  Before  use  the 
syringe  with  the  needle  should  be  boiled  for  ten  minutes 
to  sterilise  it ;  after  use  it  may  be  well  rinsed  and  again 
boiled.  The  needles  should  be  wiped  dry  and  a  wire 
inserted,  or  they  may  be  kept  in  a  bottle  of  xylol. 

Guinea-pigs  and  rabbits  are  usually  inoculated  in  the 


ANIMAL  INOCULATION  123 

thigh  or  abdomen ;  mice  in  the  dorsal  region  or  at  the 
root  of  the  tail  (dorsally),  the  hair  being  clipped,  and 
the  skin  disinfected,  but  this  is  not  generally  necessary. 
Numerous  mechanical  holders  have  been  devised  for 
animals,  but  are  not  as  a  rule  required.  Rabbits  may 
be  inoculated  intra-venously  by  one  of  the  large  veins 
in  the  ear.  The  ear  is -shaved,  and  the  skin  is  well  washed 
with  a  little  alcohol  with  vigorous  rubbing ;  the  base  of 
the  ear  is  lightly  pinched  so  as  to  obstruct  the  venous 
but  not  the  arterial  circulation,  and  render  the  vein 
prominent,  and  the  injection  is  made  with  a  small  syringe 
fitted  with  a  fine  needle,  the  needle  being  passed  into  the 
vein  towards  the  base  of  the  ear.  After  the  withdrawal 
of  the  needle  the  wound  is  compressed  for  a  little  and  may 
be  dressed  with  some  antiseptic  wool  and  collodion. 

Guinea-pigs  frequently  eat  the  carcases  of  their  dead 
companions,  so  that  the  cages  should  be  examined  twice 
daily,  and,  if  the  carcase  is  required,  it  may  be  advisable 
to  keep  each  animal  in  a  separate  cage. 

The  phenomena  occurring  after  inoculation  must  be 
noted.  Usually  these  are  not  very  obvious  in  the  rodents, 
but  loss  of  appetite,  sluggishness,  staring  coat,  convul- 
sions, etc.,  may  be  observed.  The  weight  of  the  animal 
is  a  good  index  of  what  is  happening.  If  the  infection  is 
serious,  the  weight  rapidly  falls ;  if  the  animal  is  to 
recover,  its  weight  soon  begins  to  increase  after  the  pre- 
liminary fall.  The  temperature  in  the  rectum  may  also 
be  taken,  but  is  not  so  valuable,  as  in  the  guinea-pig 
variations  occur  from  mere  handling  or  other  slight  causes. 
The  temperature  of  the  guinea-pig  averages  38-6°,  but 
varies  between  36°  and  39°  C.  (Eyre). 

The  examination  of  the  dead  animal  should  be  carried 
out  with  as  little  delay  as  possible.  For  dissection,  the 
body  should  be  pinned  out  on  the  back  on  a  board,  which 
may  stand  in  a  shallow  enamelled  iron  pan,  by  pins  or 


124  A  MANUAL  OF  BACTERIOLOGY 

nails  through  the  feet,  and  the  abdomen  well  soaked  with 
antiseptic  solution,  not  so  much  to  sterilise  the  skin  as  to 
prevent  the  hair  from  getting  into  the  incision  ;  to  obtain 
complete  sterilisation  of  the  skin,  it  is  preferable  to  clip 
or  shave  the  hair  and  then  sear  with  a  red-hot  iron. 
Knives,  forceps,  scissors,  etc.,  should  be  well  boiled  in  an 
enamelled  iron  mug  or  pie- dish,  the  water  being  kept 
boiling  during  the  progress  of  the  dissection  and  the 
instruments  rinsed  from  time  to  time  in  it.  A  little  sodium 
carbonate  may  with  advantage  be  added  to  the  water. 
A  small  enamelled  iron  fish-kettle  with  perforated  strainer 
forms  an  excellent  steriliser  for  instruments,  or  a  surgical 
instrument  steriliser  may  be  used.  An  incision  is  made 
and  the  skin  well  reflected  and  pinned  out ;  the  knife  and 
forceps  should  then  be  re-sterilised,  or  fresh  sterile  instru- 
ments taken,  for  the  deeper  incision  and  opening  the  body 
cavities ;  these  again  must  be  re-sterilised,  or  a  third  set 
of  instruments  employed  for  incising  the  organs. 

During  the  progress  of  the  dissection  the  condition  of 
the  tissues  at  the  seat  of  the  inoculation  should  be  noted, 
and  likewise  the  conditions  of  the  serous  membranes  and 
the  various  organs.  In  many  diseases  the  organism  is 
met  with  most  abundantly  in  the  spleen,  in  others  in  the 
blood,  and  in  some  at  the  seat  of  inoculation.  When  a 
systematic  examination  is  made,  film  specimens  and 
cultures  on  two  or  three  media,  aerobic  and  anaerobic, 
should  be  prepared  from  the  seat  of  inoculation,  the  spleen, 
liver,  lungs,  and  heart-blood,  and  in  some  cases  from  the 
serous  membranes,  muscles,  or  central  nervous  system 
in  addition,  the  carcase  being  in  the  intervals  covered 
with  a  bell- jar  which  has  been  rinsed  in,  or  with  filter- 
paper  moistened  with,  antiseptic  solution.  An  assistant 
is  often  useful  or  even  necessary.  The  greatest  care  must 
be  taken  to  avoid  dropping  or  splashing  or  otherwise 
disseminating  infective  material,  any  stains  being  im- 


POST-MORTEM  EXAMINATION  125 

mediately  swabbed  up  with  antiseptic  solution ;  and  the 
operator  must  exercise  every  precaution  to  prevent  the 
infection  of  himself  and  others.  It  is  convenient  to  have 
some  efficient  antiseptic  solution  near  at  hand  ;  it  may  be 
kept  in  a  large  bottle  on  a  wall  bracket  and  drawn  off  as 
required  by  a  syphon  tube  provided  with  a  tap  or  spring 
clip.  The  most  generally  used  antiseptics  are  5  per  cent, 
carbolic,  and  1-500  corrosive  sublimate,  but  2  per  cent, 
cyllin  or  kerol  or  3  per  cent,  lysol  is  cheaper  and  more 
efficient.  The  access  of  flies  to  the  carcase  must  also  be 
prevented,  as  they  might  carry  infection.  When  finished 
with,  the  carcase  should  be  efficiently  disinfected  and 
disposed  of  without  delay,  preferably  by  burning  it, 
together  with  the  board  on  which  it  has  been  pinned  out. 

If  the  carcase  be  left,  especially  in  warm  weather,  for 
even  a  few  hours  before  the  examination  is  carried  out, 
the  tissues  are  liable  to  become  invaded  and  infected  by 
organisms  from  the  respiratory  and  digestive  tracts.  In 
the  post-mortem  room,  infection  of  the  tissues  is  very 
common  ;  out  of  fifty  cases,  Symes1  found  only  seventeen 
to  be  sterile.  Ford  states  that  even  in  normal  animals, 
killed  and  immediately  examined,  bacteria  are  present 
in  70  per  cent,  of  the  internal  organs.2 

When  the  blood  of  an  animal  is  required  several  ex- 
pedients may  be  adopted.  From  large  animals,  like  the 
horse,  sheep,  and  goat,  it  may  be  obtained  by  passing 
the  needle  of  a  large  syringe  into  the  external  jugular 
vein  (which  runs  superficially  on  either  side  of  the  under 
part  of  the  neck)  and  then  aspirating  with  the  syringe. 
In  the  case  of  small  animals  not  again  needed,  the  animal 
may  be  decapitated  or  the  throat  may  be  cut,  and  the 
blood  collected  in  a  porcelain  dish  ;  but  if  a  sample  only 
is  wanted,  and  the  animal  has  to  be  further  treated,  as 

1  Lancet,  1899,  vol.  i,  p.  365. 

*  Journ,  of  Hygiene,  vol.  i,  No.  2,  1901,  p.  277. 


126  A  MANUAL  OF  BACTERIOLOGY 

in  antitoxin  work,  it  is  generally  possible  to  bleed  from  a 
superficial  vein.  The  needle  of  a  syringe  may  be  passed 
into  the  heart  of  a  guinea-pig  and  2-3  c.c.  of  blood  with- 
drawn without  injury  to  the  animal.  In  the  rabbit  blood 
may  be  obtained  by  passing  the  fine  point  of  a  piece  of 
glass  tubing,  drawn  out  and  bent  to  a  convenient  angle, 
or  the  needle  of  a  syringe,  into  one  of  the  ear  veins 
and  aspirating  the  blood  into  it.  Or  the  vein  may 
be  punctured  and  the  blood  allowed  to  drip  into  a  small 
tube. 

Blood  may  be  obtained  from  a  patient  for  the  aggluti- 
nation reaction,  for  microscopical  examination,  or  for 
culture  experiments,  by  pricking  the  finger  or  the  lobe  of 
the  ear  with  a  sterile  needle,  preferably  a  flat  one  of  the 
"  Hagedorn  "  type,  or  with  half  a  steel  pen  (nib)  or  a 
glass  point ;  for  disinfection,  the  skin  may  be  rubbed  with 
a  little  alcohol  or  ether.  After  swinging  the  arm  and 
winding  a  piece  of  rubber  tubing  round  the  finger  or 
thumb  and  pricking  1-3  c.c.  may  generally  be  obtained. 
The  blood  may  be  collected  in  a  small  test-tube,  vaccine 
tubes,  small  bulbous  tubes  (Fig.  7,  p.  52),  or  Wright's 
tubes  (Fig.  35,  p.  215). 

If  the  tube  with  contained  blood  is  sealed  in  the  flame, 
and  is  afterwards  centrifuged  to  obtain  clear  serum,  care 
should  be  taken  that  one  end  is  not  wetted  with  the  blood, 
and  this  dry  end  should  be  sealed  first  so  as  to  obtain  a 
perfect  seal,  When  centrifuging,  this  sealed  end  should 
be  placed  downwards  in  the  centrifuge  "  bucket." 

Organisms,  in  natural  infections  in  man,  are  usually 
present  only  in  small  numbers  in  the  blood,  and  for 
demonstrating  them  by  culture  methods  it  is  necessary 
to  withdraw  2-5  c.c.  from  a  superficial  or  deep  vein  by 
means  of  a  sterile  syringe  under  aseptic  conditions,  and  to 
inseminate  broth  tubes  or  agar  plates  each  with  0'5  c.c.  of 
the  blood. 


EXAMINATION  OF  LIVING  ORGANISMS        127 

Although  the  modern  methods  of  isolation  and  cultivation  have 
rendered  immense  service  to  bacteriology,  they  have  also  had  the 
effect  of  diminishing  the  attention  paid  to  the  exact  morphology 
and  biology  of  organisms.  At  the  present  time  there  is  a  tendency 
to  investigate  bacteria  en  masse  rather  than  to  study  them  as 
individual  living  forms.  As  the  late  Marshall  Ward  remarked  : 

"  The  introduction  and  gradual  specialisation  of  Koch's  method 
of  rapid  isolation  of  colonies  encouraged  the  very  dangers  they  were 
primarily  intended  to  avoid.  It  was  soon  discovered  that  pure 
cultures  could  be  obtained  so  readily  that  the  characteristic  differ- 
ences of  the  colonies  in  the  mass  could  presumably  be  made  use  of 
for  diagnostic  purposes,  and  a  school  of  bacteriologists  arose  who 
no  longer  thought  it  necessary  to  patiently  follow  the  behaviour  of 
the  single  spore  or  bacillus  under  the  microscope,  but  regarded  it 
as  sufficient  to  describe  the  form,  colour,  markings,  and  physiological 
changes  of  the  bacterial  colonies  themselves  on  and  in  different 
media,  and  were  content  to  remove  specimens  occasionally,  dry  and 
stain  them,  and  describe  their  forms  and  sizes  as  they  appeared 
under  these  conditions.  To  the  botanist,  and  from  the  point  of 
view  of  scientific  morphology,  this  mode  of  procedure  may  be 
compared  to  what  would  happen  if  we  were  to  frame  our  notions 
of  species  of  oak  or  beech  according  to  their  behaviour  in  pure 
forests,  or  of  grass  or  clover  according  to  the  appearance  of  the 
fields  and  prairies  composed  more  or  less  entirely  of  it,  or — and  this 
is  a  more  apt  comparison,  because  we  can  obtain  colonies  as  pure 
as  those  of  the  bacteriologist — of  a  mould  fungus  according  to  the 
shape,  size,  and  colour,  etc.,  of  the  patches  which  grow  on  bread, 
jam,  gelatine,  and  so  forth." 


Examination  of  Living  Organisms 

One  essential  procedure  in  the  investigation  of  an 
organism  is  its  examination  in  the  fresh  and  living  con- 
dition. This  may  be  done  by  placing  a  droplet  of  sterile 
water,  broth,  or  salt  solution  on  the  slide,  inoculating 
with  a  trace  of  the  material  or  growth,  and  covering 
with  a  cover-glass  and  examining  microscopically.  The 
action  of  stains  and  reagents  on  the  organisms  may  be 
observed  by  the  irrigation  method.  A  drop  of  the  stain 
or  reagent  (c,  Fig.  21)  is  placed  on  the  slide,  A,  just  in 


128 


A  MANUAL  OF  BACTERIOLOGY 


contact  with  one  margin  of  the  cover- glass,  B,  and  is 
drawn  through  the  preparation  by  means  of  a  small  piece 
of  filter-paper,  D,  placed  on  the  other  side,  a  torn  margin 
touching  the  film  of  fluid  at  one  edge  of  the  cover- glass. 

The  filter-paper  absorbs  the  fluid  from  under  the  cover- 
glass,  leaving  the  cells  and  other  particles  behind,  and  at 
the  same  time  the  reagent  on  the  opposite  side  flows  under 
the  cover- glass  to  take  the  place  of  the  absorbed  fluid. 
Afterwards  the  excess  of  the  reagent  or  stain  may  be 


A 


B 


FIG.  21. — Method  of  irrigation. 

washed  away  by  running  in  water  under  the  cover- glass 
in  a  like  manner.  Care  must  be  taken  that  no  fluid  gets 
on  to  the  upper  surface  of  the  cover-glass,  which  must 
always  be  kept  dry.  The  advantage  of  this  method  is 
that  it  may  be  applied  while  the  specimen  is  being  examined 
under  the  microscope,  and  the  action  of  the  reagent  on  a 
particular  cell  or  granule  can,  with  a  little  care,  be  watched. 
If  the  cells  be  large  and  it  is  desirable  to  avoid  pressure 
of  the  cover-glass,  a  fine  hair  or  bristle  may  be  so  placed 
on  the  slide  that  when  the  cover- glass  is  lowered  one 
edge  rests  on  it.  If  the  specimen  has  to  be  kept  for 
any  length  of  time,  the  film  of  fluid  will  before  long 
evaporate  and  the  preparation  become  dry.  To  prevent 
this  a  ring  of  oil  or  vaseline  may  be  painted  round  the 
margin  of  the  cover-glass  so  as  to  seal  it  to  the  slide. 

A  simple  method  for  keeping  organisms  under  examina- 
tion for  a  lengthened  period  of  time,  and  of  watching 


HANGING-DROP  PREPARATIONS  129 

their  growth  and  development,  is  by  the  use  of  hanging 
drop  preparations.  To  prepare  a  hanging  drop,  a  ring  of 
vaseline  is  painted  round  the  margin  of  the  hollow  of  a 
hollow-ground  slide  (or  other  cell,  see  below).  A  cover- 
glass  is  sterilised  by  flaming  in  the  Bunsen,  care  being 
taken  not  to  heat  sufficiently  to  melt  it.  A  droplet  of 
some  sterile  fluid  medium — water,  broth,  wort,  sugar 
solution,  etc. — is  then  placed  in  the  centre  of  the  cover- 
glass  with  a  sterile  platinum  loop.  This  droplet  is  then 
inoculated  with  the  organism  which  is  to  be  observed, 
care  being  taken  not  to  add  too  many  organisms — a  few 


FIG.  22. — Hanging-drop  preparation. 

isolated  organisms  and  small  groups  in  each  field  is  what 
should  be  aimed  at.  The  vaselined  cell  is  now  taken  and 
turned  over,  so  that  the  ring  of  vaseline  is  downwards, 
and  is  then  applied  to  the  cover-glass,  in  such  a  way  that 
the  droplet  is  situated  in  the  middle  of  the  hollow,  but  not 
touching  the  slide  at  any  point.  The  cover-glass  adheres 
to  the  slide  by  means  of  the  vaseline,  and  on  quickly 
inverting  the  whole,  so  that  the  fluid  has  no  time  to  run, 
it  will  be  found  that  the  droplet  is  hanging  from  the 
under  surface  of  the  cover- glass  in  a  cell  which  is  hermeti- 
cally sealed  by  the  vaseline,  and  evaporation  is  thus 
rendered  impossible  (Fig.  22).  Such  a  preparation,  in 
fact,  can  be  kept  for  a  week  or  ten  days  in  a  warm  incubator 
without  drying  up.  Great  care  must  be  exercised  in 
examining  a  hanging-drop  specimen  microscopically, 
especially  with  the  immersion  lenses,  for  the  slightest 
pressure  breaks  the  unsupported  cover-glass.  It  often 
saves  time  first  to  centre  the  drop  with  the  low  power 
before  examining  with  the  immersion  lens ;  an  ink  or 
pencil  dot  at  the  margin  of  the  drop  aids  focussing.  The 

9 


130  A  MANUAL  OF  BACTERIOLOGY 

light  must  be  diminished  by  closing  the  diaphragm,  lowering 
the  condenser,  etc.  (p.  132),  and  artificial  light  is  generally 
preferable  to  daylight.  The  central  parts  of  the  drop 
only  should  be  examined,  not  the  margin. 

Instead  of  hollow  slides,  various  devices  may  be  em- 
ployed to  form  the  cell.  Metal,  glass,  or  vulcanite  rings, 
or  rings  cut  out  of  thin  sheet  lead,  tin-foil,  cardboard,  or 
two  or  three  thicknesses  of  paper  or  filter-paper  may  be 
cemented  on  to  slides  with  vaseline,  Hollis's  glue,  gold 
size,  or  Canada  balsam,  or  a  thick  ring  of  vaseline,  or 
paraffin,  or  plasticine  may  be  used. 

The  only  certain  method  for  ascertaining  whether  an 
organism  is  motile  or  not — often  an  important  clue  to  its 
identification — is  by  the  use  of  hanging-drops.  Actively 
motile  organisms  may  frequently  assume  a  non-motile 
resting  stage,  although  still  alive,  and  various  factors 
may  bring  about  this  condition,  such  as  old  age,  exhaustion 
of  nutriment,  excessive  heat  or  cold,  electric  shocks,  and 
the  like.  The  absence  of  movement  of  an  organism  in  a 
specimen  prepared  from  an  ordinary  culture,  particularly 
if  more  than  a  day  or  two  old,  does  not  necessarily  prove 
that  it  is  non-motile.  A  hanging-drop  should  be  prepared 
with  a  nutrient  medium  (the  best,  perhaps,  is  glucose 
broth)  and  placed  under  conditions  of  temperature,  etc., 
favourable  to  the  growth  of  the  organism,  and  examined 
after  an  interval  of  an  hour  or  so,  or  better  still  at  intervals 
of  half  an  hour  for  three  or  four  hours.  In  this  time  the 
old  cells  will  revivify,  and  new  ones  will  have  been  pro- 
duced, and  if  the  organism  be  a  motile  one,  more  or  less 
active  movement  of  some  of  the  cells  is  almost  sure  to  be 
observed.  It  is  necessary  to  beware  of  two  fallacies  in 
connection  with  motility — not  to  mistake  for  it  the  so-called 
Brownian  movement,  which  is  a  vibratory  one  back- 
wards and  forwards  about  one  point,  and  common  to 
all  fine  particles  suspended  in  a  fluid ;  and  not  to  be 


INTERLAMELLAR  FILMS  131 

misled  by  a  flotation  of  the  cells  due  to  currents  set  up 
in  the  fluid  from  some  cause  or  other— all  the  particles 
then  tending  to  move  in  the  same  direction. 

Another  purpose  for  which  the  hanging-drop  cultivation 
may  be  employed  is  that  of  obtaining  a  permanent  record 
of  the  various  phases  through  which  an  organism  may  pass 
during  its  development.  If  a  number  of  these  cultivations 
be  made,  say  twenty,  in  an  exactly  similar  manner,  and 
afterwards  kept  under  identical  conditions,  and  if  at  the 
end  of  every  half-hour  one  of  the  preparations  be  taken, 
its  cover-glass  carefully  removed,  and  the  droplet  dried 
and  stained,  a  permanent  record  of  the  life-history  of  the 
organism  is  obtained  extending  over  ten  hours. 

Various  more  elaborate  forms  of  cells  for  hanging-drop 
preparations  can  be  obtained,  some  being  provided  with 
inlet  and  exit  tubes  for  the  passage  of  various  gases.  For 
anaerobic  preparations  cells  are  made  having  a  groove 
at  the  bottom  into  which  a  mixture  of  pyrogallic  acid 
and  potash  is  introduced. 

The  observation  of  hanging-drop  cultivations  at 
blood-heat  can  be  carried  out  on  some  form  of  warm 
stage. 

Interlamellar  films.1 — Another  method  of  investigating  the  life- 
history  of  organisms,  especially  moulds  and  protozoa,  is  by  means 
of  interlamellar  films.  A  glass  slide  1|  by  3  in.  is  sterilised  in  the 
Bunsen  flame,  and  while  hot  three  small  drops  of  sealing-wax  are 
placed  on  it,  so  arranged  that  they  form  the  apices  of  an  equilateral 
triangle,  the  side  of  which  measures  about  one  inch,  and  a  drop 
of  sterile  nutrient  medium  is  deposited  between  them.  A  cover- 
glass  of  about  1^  in.  in  diameter  is  then  sterilised  in  the  Bunsen 
flame,  a  droplet  of  a  suitable  nutrient  medium  is  placed  upon  it 
and  inoculated  with  the  organism  to  be  observed,  and  the  pre- 
pared cover-glass  is  picked  up  with  sterilised  forceps,  inverted, 
and  lowered  on  to  the  slide.  The  nutrient  medium  is  thus  contained 
between  the  slide  and  the  cover-glass,  and  by  using  a  hot  wire, 
and  so  softening  the  sealing-wax,  it  can  be  spread  out  to  form  as 

1  Delepine,  Lancet,  1891,  vol.  i,  June  13. 


132  A  MANUAL  OF  BACTERIOLOGY 

thin  a  layer  as  desired.     The  preparation  is  kept  in  a  moist  chamber 
to  prevent  evaporation,  and  can  be  studied  when  required. 


The  Microscope 

A  bacteriological  microscope  is  generally  of  the  mono- 
cular form,  and  should  be  provided  with  a  rack-and-pinion 
coarse  adjustment  and  an  efficient  fine  adjustment.  The 
stage,  preferably  of  vulcanite,  should  be  large  and  roomy 
and  quite  plain,  with  two  or  more  holes  at  its  margin  to 
receive  spring  clips  for  fixing  the  slide.  For  the  ordinary 
examination  of  specimens  a  mechanical  stage  is  not  needed  ; 
in  fact  it  hampers  that  freedom  of  manipulation  which 
is  so  useful  for  the  rapid  examination  of  a  specimen.  For 
some  purposes  a  mechanical  stage  is  very  useful,  and  for  a 
critical  survey  of  the  whole  of  a  specimen,  e.g.  a  blood- 
film,  it  is  essential.  A  detachable  form  is  to  be  preferred 
(Fig.  23),  so  that,  if  required,  the  stage  may  be  free  for 
the  examination  of  plate  cultivations,  etc. 

New  forms  of  binocular  microscopes  have  recently  been 
introduced  by  Messrs.  Beck  and  by  Messrs.  Leitz  which 
possess  marked  advantages  over  the  monocular  instru- 
ment. 

A  sub- stage  condenser  is  essential  for  all  work  in  which 
high  powers  are  employed,  and  also  enhances  the  value  of 
low  powers.  It  consists  of  a  system  of  lenses  below  the 
stage,  by  means  of  which  the  light  is  concentrated  on 
the  object.  It  should  have  a  rack-and-pinion,  or  a  screw, 
adjustment  for  focussing,  and  be  provided  with  some 
form  of  diaphragm  for  modifying  the  light,  preferably 
an  "  iris."  The  condenser  must  be  centred — that  is, 
adjusted  so  that  its  optical  axis  corresponds  with  the 
optical  axis  of  the  objective  ;  and  for  this  purpose  it  ought 
to  be  provided  with  two  lateral  screws  working  at  right 
angles  to  each  other,  by  means  of  which  its  position 


THE  MICROSCOPE  133 

relative  to  the  optical  axis  can  be  altered.  In  order  to 
centre,  a  diaphragm  with  small  aperture  is  used,  and  the 
hole  in  the  diaphragm  is  focussed  with  a  low  power  ;  then, 
by  means  of  the  lateral  screws,  this  hole  is  brought  into 
the  centre  of  the  field.  Below  the  sub-stage  condenser 
a  mirror  with  concave  and  plane  surfaces  should  be  fitted, 
the  plane  surface  being  used  with  the  condenser,  as  a 


FIG.  23. — Swift's  detachable  mechanical  stage. 

general  rule.  The  concave  mirror  may  be  used  for  illumi- 
nation with  low-power  objectives,  the  condenser  being 
detached  or  swung  out  of  position.  The  necessity  for 
careful  illumination  must  be  insisted  upon ;  in  fact,  to 
obtain  the  best  results  the  light  should  be  readjusted  for 
every  specimen  by  mirror,  diaphragm,  and  condenser, 
i.e.  "  critical  "  illumination  should  be  aimed  at.  A  good 
specimen  may  be  utterly  spoilt,  visually,  by  faulty  illumi- 
nation ;  while  an  indifferent  one  may  be  made  to  look 
passable  by  proper  illumination.  In  the  examination  of 
micro-organisms  in  the  fresh  or  living  and  unstained 
condition,  it  is  necessary,  as  a  rule,  to  diminish  the  light 
by  means  of  a  small  diaphragm,  or  by  racking  down  the 
condenser,  or  by  both  ;  while  for  stained  or  opaque  objects 


134  A  MANUAL  OF  BACTERIOLOGY 

the  full  aperture  of  the  diaphragm,  or  thereabouts,  may 
generally  be  employed.  It  must  be  remembered,  however, 
that  the  resolving  power  of  a  lens  (see  below)  is  diminished 
by  closing  the  diaphragm  and  by  throwing  the  condenser 
out  of  focus  ;  the  illumination  then  becomes  "  non-critical." 
For  fine  work,  if  the  illumination  is  too  intense,  this  should 
be  diminished  by  diminishing  the  source  of  light  or  by 
interposing  a  coloured  screen,  such  as  Gifford's,  which 
consists  of  a  cell  containing  a  solution  of  malachite  green 
in  which  is  inserted  a  piece  of  green  signal  glass.  Coloured 
glass  may  also  be  interposed.  The  microscopist  should 
accustom  himself  to  examine  specimens  both  by  daylight 
and  by  artificial  light ;  hanging-drop  specimens  are 
usually  best  seen  with  the  latter.  For  artificial  light, 
probably  nothing  surpasses  a  paraffin  lamp  with  flat 
wick,  the  edge  of  the  flame  being  always  used,  while  to 
obtain  the  best  results  the  mirror  should  be  removed,  and 
the  flame  used  direct  by  elevating  and  tilting  the  micro- 
scope somewhat.  For  the  finest  work,  daylight  illumina- 
tion is  inadmissible.  An  admirable  form  of  electric  lamp 
is  the  "  Barnard,"  made  by  Messrs.  Swift  and  Son,  the 
source  of  illumination  being  a  Nernst  lamp.  For  ordinary 
routine  work,  an  incandescent  carbon  or  metal  filament 
electric  lamp,  a  Nernst  lamp,  or  an  argand  or  incan- 
descent gas  burner  may  be  used.  Various  devices  have 
been  introduced  for  the  employment  of  monochromatic 
illumination,  e.g.  the  quartz  mercury  vapour  lamp  by 
Barnard. 

With  the  filament,  Nernst,  or  incandescent  gas,  lamps, 
the  image  of  the  filament  or  mantle  is  troublesome  when 
the  condenser  is  in  focus  ;  this  may  be  obviated  to  some 
extent  by  the  use  of  frosted  bulbs  or  by  interposing  a 
screen  of  fine  ground  glass,  by  the  use  of  Gordon's  glass  rod 
illuminator,  or  by  interposing  a  spherical  flask  filled  with 
water  or  dilute  copper  sulphate  solution.  Incandescent 


THE  MICROSCOPE  135 

bulbs  may  be  frosted  by  dipping  in  a  15  per  cent,  solution 
of  caustic  soda  and  allowing  to  dry. 

Two  eyepieces  are  sufficient,  and  the  lower-power  ones 
are  to  be  preferred,  such  as  the  B  and  c  of  the  English, 
or  the  2  and  3  of  the  Continental,  makers.  Although 
increased  magnification  can  be  obtained  by  the  use  of  a 
high-power  eyepiece,  it  is  at  the  expense  of  definition, 
the  image  losing  its  sharpness,  because  the  eyepiece  mag- 
nifies the  image  formed  by  the  objective,  and  any  imper- 
fections in  the  latter  are  made  more  apparent,  so  that  the 
use  of  very  high  eyepieces  is  not  to  be  recommended, 
except  with  the  finest  lenses  ;  moreover,  as  will  be  pointed 
out  later,  it  is  useless  to  increase  the  amplification  beyond 
a  certain  point. 

With  regard  to  the  length  of  the  tube  of  the  microscope, 
this  differs  in  the  English  and  Continental  systems.  The 
standard  English  tube-length  is  8-75  in.,  the  Continental 
is  6-3  in.,  and  is  usually  adopted,  but  the  longer  tube  gives 
greater  amplification.  The  tube  of  the  microscope  is 
generally  provided  with  an  inner,  or  draw-tube,  by  means 
of  which  its  length  can  be  nearly  doubled  ;  this  gives 
increased  amplification,  but  at  the  expense  of  definition, 
at  least  with  the  higher  powers  which  are  corrected  or 
adjusted  for  a  definite  tube-length. 

The  lenses  or  objectives  must  next  be  considered. 

For  powers  higher  than  the  J-in.,  or  thereabouts,  it  is 
advisable,  for  many  reasons,  to  employ  the  immersion 
system  of  objectives.  With  these  lenses  a  drop  either  of 
water,  in  the  water-immersion  system,  or  of  cedar  oil,  in 
the  oil- immersion  one,  is  placed  on  the  cover- glass,  and 
the  objective  is  racked  down  so  that  its  front  lens  touches 
and  is  immersed  in  either  the  water  or  oil,  as  the  case 
may  be.  It  is  a  good  plan  then  to  raise  the  objective  very 
slightly  by  means  of  the  coarse  adjustment,  still,  however, 
keeping  it  in  contact  with  the  drop  of  water  or  oil.  The 


136 


A  MANUAL  OF  BACTERIOLOGY 


observer  then,  looking  down  the  microscope,  very  cautiously 
and  gradually  racks  down  again  with  the  coarse  adjust- 
ment until  the  object  comes  into  view,  and  finishes  the 
focussing  with  the  fine  adjustment.  The  fine  adjustment 
should  only  be  used  after  the  object  has  been  brought  into 
view  by  means  of  the  coarse  adjustment.  After  the 
examination  has  been  concluded  for  the  day,  the  lens 

Cl 


y 


FIG.  24. — Diagram  to  illustrate  the  refraction  of  light. 

should  be  carefully  wiped  with  a  soft  rag,  or  preferably 
with  a  piece  of  soft  Japanese  paper,  to  remove  the  water  or 
oil.  If  the  oil  should  happen  to  dry  on  the  lens,  it  may  be 
removed  by  wiping  with  a  soft  rag  or  Japanese  paper  moist- 
ened with  xylol,  quickly  drying  with  another  rag  or  paper. 
Instead  of  cedar-oil,  a  liquid  paraffin  has  also  been  used. 

The  T^  in.  (2  mm.)  oil-immersion  lens  is  the  one  usually 
selected.  It  combines  sufficient  magnification  for  most 
purposes  with  adequate  working  distance  for  convenience 
in  using.  If  expense  is  not  an  object,  the  Zeiss  J  in. 
(3  mm.)  apochromatic  oil-immersion  lens  is  a  very  fine  one 
for  general  use.  By  means  of  the  compensating  oculars 


THE  IMMERSION  SYSTEM 


137 


sufficient  magnification  can  be  obtained,  while  the  working 
distance  is  greater,  the  field  is  larger,  and  the  penetrative 
power  is  greater  than  with  the  ^  in.  lens. 

The  immersion  system  of  objectives  has  many  advantages  :  the 
loss  of  light  is  less,  the  distance  between  the  cover-glass  and  the 
front  of  the  objective — the  working  distance,  as  it  is  termed — is 
greater,  and  more  can  be  seen  with  an  immersion  lens  than  with 


S-. 


FIG.  25. — Diagram  to  illustrate  tlje  course  of  rays  of  light 
through  an  objective. 

a  dry  lens  of  equal  magnifying  power.  This  can  be  best  illustrated 
by  means  of  two  simple  diagrams. 

In  Fig.  24  let  cd  represent  the  surface  of  a  fluid,  either  water  or 
oil,  and  let  ab  be  drawn  perpendicular  to  this  surface,  and  cutting 
it  at  y.  Let  ry  represent  a  ray  of  light  proceeding  from  a  rarer 
medium,  such  as  air,  into  a  denser  one,  water  or  oil.  As  is  well 
known,  this  ray  when  it  enters  either  the  water  or  the  oil  does  not 
continue  in  the  same  direction,  but  is  "  refracted  "  or  bent  nearer 
the  perpendicular  ab,  the  bending  being  more  marked  with  oil 
than  with  water.  Thus  we  may  suppose  that  the  direction  of  the 
ray  in  water  would  be  represented  by  the  line  yw,  and  in  oil  by  the 
dotted  line  yo.  Conversely,  a  ray  of  light  proceeding  from  a 
denser  medium  into  a  rarer  is  bent  away  from  the  perpendicular, 
and  the  rays  wy  in  water,  and  oy  in  oil,  would,  on  emerging  into 
air,  proceed  in  the  direction  yr. 

In  Fig.  25  (which  for  convenience  is  drawn  somewhat  out  of 


138  A  MANUAL  OF  BACTERIOLOGY 

proportion)  let  s  represent  an  ordinary  glass  micro-slide,  x  a  layer 
of  Canada  balsam  in  which  the  object  is  mounted,  and  covered 
with  the  cover-glass  G,  while  L  is  the  objective  with  its  front  lens. 
Let  the  object  be  illuminated  by  the  ray  of  light  Yy  ;  this  on  enter- 
ing the  glass  of  the  slide  and  the  Canada  balsam  will  be  refracted 
or  bent  nearer  the  perpendicular  and  will  proceed  in  the  direction 
yt.  Canada  balsam,  and  also  cedar  oil,  produce  about  the  same 
amount  of  "  refraction,"  or  bending  of  a  ray  of  light,  as  crown 
glass,  and  hence  these  three  substances — crown  glass,  Canada 
balsam,  cedar  oil — are  said  to  have  the  same  "  refractive  index," 
and,  consequently,  the  glass  of  the  slide,  the  Canada  balsam,  and 
the  cover-glass  act  as  one  homogeneous  medium,  and  the  line  yt 
is  a  straight  one.  In  the  first  place,  let  us  suppose  that  the 
objective  L  is  a  dry  one,  having  a  layer  of  air  between  its  front  lens 
and  the  cover-glass  ;  then  the  ray  of  light,  on  emerging  from  the 
cover-glass  into  the  air,  is  now  bent  away  from  the  perpendicular 
and  pursues  a  direction  practically  parallel  to  its  former  one, 
represented  by  the  line  tw,  and  misses  the  lens  altogether — the 
lens  is  unable  to  take  it  up.  If,  however,  we  suppose  that  our 
objective  is  an  oil-immersion  one,  and  that  a  drop  of  cedar  oil 
takes  the  place  of  the  layer  of  air  between  the  cover-glass  and  the 
front  lens  in  the  foregoing  example,  then  the  glass  slide,  Canada 
balsam,  cover-glass,  cedar  oil,  and  the  front  lens  of  the  objective 
form  practically  one  medium  ;  they  all  have  the  same  refractive 
index  and  produce  the  same  amount  of  refraction  or  bending  of  a 
ray  of  light.  Therefore  the  direction  of  the  ray  forms  a  straight 
line  in  all  these,  and  the  ray  passes  into  the  objective  as  is  repre- 
sented by  the  broken  line  t — v.  More  important  still,  however,  is 
that  which  happens  to  rays  which  fall  on  the  slide  at  a  very  oblique 
angle.  In  the  same  figure  (Fig.  25)  let  ef  represent  such  a  ray  ; 
on  entering  the  slide  it  will  be  refracted,  and  its  passage  through 
the  slide,  balsam,  and  cover-glass  may  be  represented  by  fk.  As 
before,  let  us  suppose  that  in  the  first  place  our  objective  is  a  dry 
one,  and  that  we  have  a  layer  of  air  between  the  cover-glass  and  its 
front  lens.  In  this  case,  if  the  angle  which  fk  makes  with  the 
perpendicular  is  greater  than  about  39°  or  40°,  the  ray,  instead  of 
emerging  from  the  cover-glass  into  the  layer  of  air,  is  totally  reflected 
by  the  cover-glass  and  pursues  a  course  roughly  represented  by  kr, 
so  that  it  never  enters  the  objective.  If,  however,  we  employ  an 
oil-immersion  objective,  with  oil  instead  of  air  between  the  cover- 
glass  and  its  front  lens,  then,  as  before,  the  slide,  balsam,  cover- 
glass,  oil,  and  front  lens  of  the  objective  form  practically  one 
homogeneous  whole,  and  the  ray  efk,  instead  of  being  totally 


DARK  GROUND  ILLUMINATION  139 

reflected,  continues  its  course  in  a  straight  line,  and  is  taken  up  by^ 
the  objective,  as  is  represented  by  the  dotted  line  k — v'.     Hence  we 
see  that  the  same  rays  which  are  unable  to  enter  a  dry  objective 
are  admitted  by  an  oil-immersion  one,  and  that  an  oil-immersion 
lens  can  take  up  rays  which  fall  on  the  slide  at  a  very  oblique  angle. 

In  order  that  these  oblique  rays  may  be  present,  ready  to  be 
taken  up  by  the  oil-immersion  objective,  it  is  necessary  to  employ 
a  sub-stage  condenser.  It  is  only  by  means  of  a  sub-stage  condenser 
that  a  "  wide -angled  cone  of  rays,"  as  it  is  termed,  is  obtained. 
Hence  to  make  full  use  of  an  oil-immersion  objective — to  "  get 
most  out  of  it  " — it  is  absolutely  essential  to  employ  a  sub-stage 
condenser,  and  for  the  finest  work  a  special  "  oil -immersion  con- 
denser "  is  employed.  It  will  be  obvious  also  that  although  a 
water-immersion  objective  admits  more  rays  than  a  dry  one,  it  does 
not  admit  so  many  as  an  oil-immersion.  It  must  be  pointed  out, 
however,  that  Canada  balsam,  or  some  medium  having  the  same  or 
a  higher  refractive  index,  must  be  used  for  mounting  to  obtain  the 
full  advantage  of  the  oil-immersion  system.  The  oil-immersion 
can  of  course  be  used  for  examining  objects  mounted  in  water,  etc., 
cedar  oil  being  still  used  between  the  cover-glass  and  the  lens.  It 
is  to  be  noted  that  a  dry  objective  cannot  be  used  as  an  immersion 
one,  nor  an  immersion  objective  dry,  as  the  construction  differs  in 
the  two  cases. 

Of  late  "  dark  ground  illumination  "  has  been  much  employed, 
particularly  for  the  examination  of  living  objects.  In  this  special 
condensers  are  used,  the  central  rays  passing  through  which  are 
"  stopped  out,"  so  that  the  object  is  illuminated  only  by  very 
oblique  rays  and  appears  white  on  a  dark  background.  A  dry  lens 
is  used,  or  if  an  oil-immersion  one,  a  stop  must  be  introduced  to 
reduce  its  aperture,  and  slides  and  cover-glasses  of  special  thickness 
together  with  brilliant  illumination  are  necessary. 

The  lenses  in  the  objective  are  formed  by  cementing 
together  different  kinds  of  glass  in  order  to  correct  for 
"  spherical  "  and  for  "  chromatic  "  aberration.  The  rays 
passing  through  the  margin  and  the  centre  of  a  simple 
lens  are  not  focussed  at  the  same  point,  and  a  distorted 
image  is  the  result ;  this  is  known  as  "  spherical  aberra- 
tion," while  the  violet  and  red  ends  of  the  spectrum, 
being  of  different  refrangibility,  and  a  simple  lens  acting 
like  a  prism,  coloured  fringes  are  observed  ;  this  is  termed 


140  A  MANUAL  OF  BACTERIOLOGY 

"chromatic  aberration."  The  apochromatic  system  of 
objectives  and  eyepieces  has  these  defects  very  perfectly 
corrected  by  the  use  of  special  glass  and  fluorite,  correction 
being  partly  effected  in  the  objective,  and  this  is  com- 
pleted by  combination  with  the  special  eyepieces.  The 
latter,  termed  "  compensating  oculars,"  are  therefore 
essential  for  perfect  correction  with  apochromatic  objec- 
tives, but  can  also  be  used  with  ordinary  lenses.  For 
photographic  purposes  apochromatic  lenses  are  far  superior 
to  achromatic  ones.  Apochromatic  objectives  are,  how- 
ever, expensive,  and  though  advantageous  are  not  really 
necessary  for  ordinary  bacteriological  work. 

In  consequence  of  certain  optical  principles,  the 
"  diffraction  "  theory,  for  details  of  which  the  reader  must 
refer  elsewhere,1  it  is  useless  to  increase  the  magnifying 
power  of  objectives  beyond  a  certain  point ;  for,  although 
the  object  viewed  appears  larger,  no  more  details  of  structure 
can  be  made  out. 

The  use  of  the  immersion  system  increases  the  "  re- 
solving power,"  or  the  amount  of  detail  which  can  be 
seen.  Thus,  if  a  number  of  fine  equidistant  parallel  lines 
be  ruled  on  a  glass  plate,  it  is  impossible  to  see  with  a  dry 
lens,  using  white  light,  more  than  about  90,000  lines  to 
the  inch  as  isolated  lines.  If  more  are  ruled  they  will 
not  appear,  and  practically  nothing  is  visible.  With  a 
water-immersion  objective  it  is  possible  to  see  about 
120,000  lines  to  the  inch,  and  with  an  oil-immersion  as 
many  as  146,000  lines  to  the  inch,  as  separate  lines — a 
clear  gain  in  resolving  power  in  the  latter  case  of  about 
one  half  over  a  dry  lens.2  As  it  is  necessary,  in  order  to 
see  such  fine  structures  as  lines  ruled  50,000  or  more  to 
the  inch  must  be,  to  have  considerable  amplification  in 

1  See  Carpenter  on  the  Microscope,  edited  by  Dallinger.     (Churchill.) 

2  These  figures  refer  to  lenses  having  a  numerical  aperture  of  1-0 
(dry),  1-33  (water),  and  1-4  (oil). 


ULTRA-MICROSCOPIC  ORGANISMS          141 

addition  to  resolving  power,  not  much  is  gained,  in 
ordinary  work  at  any  rate,  by  adopting  the  immersion 
system  for  the  lower  power  objectives,  such  as  the  g-in. 

By  the  physical  theory  of  microscopical  visibility,  it  can  be 
shown  that  objects  having  a  diameter  of  less  than  about  0-16  p 
cannot  be  seen  with  the  best  optical  appliances.  If,  then,  a  micro- 
organism is  less  in  size  than  this  it  could  not  be  seen  microscopically, 
and  this  fact  may  explain  why  it  is  that  in  certain  undoubted 
infective  diseases  no  micro-organism  has  yet  been  isolated.  Of  the 
existence  of  such  "  ultra-microscopic  "  organisms  we  have  proof. 
The  finest  porcelain  filters,  such  as  the  Chamberland  B,  do  not  allow 
visible  particles  to  pass  through,  yet  in  several  instances,  if  the 
infective  material  be  filtered  through  such  a  filter,  the  filtrate  is 
still  infective.  This  is  the  case  with  the  blood-serum  in  yellow 
fever,  Cape  horse  sickness,  dog  distemper,  hog  cholera,  and  swine 
fever,  in  bird  and  cattle  plagues,  and  with  the  juice  of  bird  mollus- 
cum.  The  organism  of  cattle  pleuro-pneumonia  is  just  on  the  limit 
of  visibility.  The  rabic  and  vaccine  viruses  also  seem  capable  of 
passing  through  a  Berkfeld  V.  These  experiments  do  not  neces- 
sarily prove  that  the  organism  in  all  stages  is  invisible. l  Siedentopf 
and  Zsigmondy  have  devised  a  method  whereby  ultra-microscopical 
particles  may  be  rendered  visible,  but  inasmuch  as  they  appear 
merely  as  luminous  points,  it  is  questionable  whether  the  method 
will  be  of  great  service  in  bacteriology.  Some  thirty  ultra - 
microscopic  viruses  are  now  known,  including,  in  addition  to  those 
mentioned  above,  those  of  anterior  poliomyelitis,  measles,  mol- 
luscum,  and  trachoma. 

There  is  no  real  necessity  in  bacteriological  work  for  the 
immersion  objective  to  be  provided  with  a  "  correction 
collar."  The  "  correction  collar  "  is  an  additional  screw 
in  the  objective  by  means  of  which  the  distance  between 
some  of  its  constituent  lenses  can  be  altered  to  "  correct  " 
for  varying  thicknesses  of  cover- glass,  etc.,  and  though 
necessary  with  the  higher  power  dry  lenses,  it  is  theo- 
retically unnecessary  with  the  immersion  system.  Never- 
theless, as  slight  variations  do  occur  in  the  various  media, 

1  See  Roux,  Bull,  de  VInst.  Past.,  vol.  i,  1903,  pp.  1  and  49.  Rem- 
linger,  ibid.  vol.  iv,  1906,  pp.  337  and  385;  Trans.  XVIIth  Internal. 
Cong.  Med.  1913,  Sect.  IV,  Pt.  I,  pp.  35  (Loffler)  and  49  (McFadyean). 


142  A  MANUAL  OF  BACTERIOLOGY 

glass,  oil,  etc.,  and  they  may  not  form  a  truly  homogeneous 
whole,  for  the  finest  work  the  correction  collar  is  still 
desirable.  So  much  for  the  high-power  objectives.  As 
regards  the  lower  powers,  which,  of  course,  are  dry,  a 
f-in.  and  a  |-in.  are  generally  selected.  The  f-in.  is  a 
more  serviceable  lens  than  the  1-in.  which  is  often  recom- 
mended. A  very  useful  accessory  is  a  "  double "  or 
"  triple  nosepiece."  This  consists  of  a  light  metal  frame- 
work, which  is  attached  to  the  lower  end  of  the  tube  of 
the  microscope,  on  to  which  two  or  three  objectives  can 
be  screwed.  The  framework  can  be  rotated,  thus  bringing 
each  objective  in  succession  into  the  optical  axis  of  the 
instrument,  and  the  necessity  for  unscrewing  and  screwing 
on  each  time  an  objective  is  changed  is  obviated.  A 
microscope  such  as  described,  with  sub-stage  condenser, 
two  eyepieces,  a  f-in.  and  a  J-in.  dry  and  a  ^--in.  oil- 
immersion  objectives,  triple  nosepiece,  etc.,  complete  in 
case,  can  be  obtained  for  about  £15,  and  it  is  well  to  add 
another  sovereign  or  two  for  superior  finish.  Both  British 
and  Continental  firms  supply  microscopes  arranged  as 
indicated,  and  in  this  department  the  English  makers 
hold  their  own. 

The  measurement  of  micro-organisms  is  carried  out  by 
means  of  a  stage  micrometer,  alone,  or  in  combination 
with  an  eyepiece  micrometer.  The  former  consists  of 
a  scale  of  tenths  and  hundredths  of  a  millimetre  or 
hundredths  and  thousandths  of  an  inch  ruled  in  fine  lines 
on  a  glass  plate,  by  means  of  which  the  measurements 
can  be  made  by  focussing  the  scale  under  the  microscope. 
The  stage  micrometer  is  placed  in  position  on  the  stage 
and  the  scale  is  focussed  with  the  particular  ocular, 
objective,  and  tube  length  which  are  to  be  used.  A 
drawing  of  the  scale  is  made  with  a  camera  lucida  ;  the 
micrometer  is  then  removed  and  the  object  placed  in 
position  and  a  second  drawing  is  made  of  the  object  on  the 


MEASUREMENT  OF  MICRO-ORGANISMS      143 

scale  already  drawn.  A  simpler  and  less  expensive  arrange- 
ment is  to  make  use  of  a  disc  of  glass  ruled  with  equi- 
distant fine  lines,  which  can  be  placed  in  the  eyepiece  by 
unscrewing  the  top  lens  and  dropping  it  on  the  diaphragm 
below.  The  value  of  the  divisions  in  the  eyepiece  scale 
is  first  ascertained  by  means  of  the  stage  micrometer. 
The  stage  micrometer  is  then  removed  and  the  object  to 
be  measured  put  in  its  place,  and  its  dimensions  are 
determined  by  means  of  the  eyepiece  scale.  With  the 
eyepiece  micrometer,  the  value  of  the  divisions  is  first 
ascertained  by  means  of  the  stage  micrometer,  which  is 
then  replaced  by  the  object.  If  the  objective  or  the  eye- 
piece be  changed  the  value  of  the  divisions  of  the  eyepiece 
scale  in  both  cases  will  be  altered,  and  must  again  be 
determined  by  means  of  the  stage  micrometer.  The  unit 
for  microscopical  measurement  is  the  micron  (sometimes 
erroneously  termed  a  micro-millimetre),  which  measures 
one  thousandth  of  a  millimetre,  or  approximately  0^375- 
of  an  inch,  and  is  designated  by  the  sign  /m. 

If  a  micrometer  is  not  available,  rough  measurements 
may  be  carried  out  by  comparison  with  a  red  blood- 
corpuscle.  The  majority  of  the  red  corpuscles  of  normal 
human  blood  measure  7-5  JUL  in  diameter. 


CHAPTER  V 

INFECTION— VEGETABLE  AND  ANIMAL  PARASITES— 
THE  INFECTIVE  PROCESS  —  ANTI-BODIES  —  ANTI- 
SERA  AND  ANTITOXINS— IMMUNITY 

Infection 

BY  the  term  INFECTION  is  meant  the  invasion  of  the  living 
tissues  by  living  micro-organisms  which  grow  and  multiply 
at  the  expense  of  the  host.  A  disease  produced  by  the 
growth  and  multiplication  of  micro-organisms  is  termed 
an  infective  disease,  and  is  transmissible  in  most  instances 
by  inoculation.  If  the  micro-organisms  are  from  time  to 
time  discharged  from  the  body  of  the  host,  either  with 
the  excreta,  secretions,  desquamated  particles,  or  in  some 
other  way,  the  disease  becomes  infectious  or  contagious, 
according  to  the  ease  with  which  another  individual 
becomes  infected,  and  the  material  which  conveys  the 
infection  is  often  termed  the  contagion.  Thus,  in  scarlatina 
and  smallpox  the  contagion  is  very  readily  conveyed  from 
person  to  person  even  for  a  distance  through  the  air,  and 
these  are  infectious  diseases.  Ringworm  and  syphilis, 
as  a  rule,  require  more  or  less  close  contact  for  infection 
to  take  place,  and  these  are,  therefore,  contagious  diseases  ; 
while  malaria  is  neither  infectious  nor  contagious,  since 
persons  in  the  neighbourhood  never  directly  contract  the 
disease,  though  it  can  be  conveyed  by  inoculation,  and 
it  is  therefore  infective  only.  But  the  distinction  between 
infectious  and  contagious  is  mainly  one  of  degree,  and  these 

144 


INFECTION  145 

terms  have  now  to  a  laTge  extent  been  discarded.  Ex- 
cluding individual  susceptibility,  the  relative  infectivity 
of  a  disease  probably  depends  on  three  factors  :  (1)  the 
contagion  is  freely  given  off  aerially  and  is  not  destroyed 
thereby ;  (2)  the  contagion  gains  access  by  the  respira- 
tory tract ;  and  (3)  the  relative  virulence  of  the  contagion  ; 
in  some  instances  the  smallest  amount  of  the  contagion 
is  sufficient  to  infect.  If  the  contagion  can  gain  access 
only  through  a  wound  or  the  digestive  tract,  the  chances 
of  infection  may  be  largely  reduced.  In  certain  instances 
infection  is  conveyed  by  an  intermediary,  e.g.  the  mosquito 
in  malaria,  and  in  such  cases  infectivity  will  obviously 
depend  on  the  presence  and  abundance  of  the  intermediary. 
Infection  is  manifestly  a  part  of  the  whole  subject  of 
parasitism,  which  includes  the  animal  and  vegetable 
parasites  which  develop  in  the  animal  body.  If,  however, 
the  subject  of  parasitism  is  considered  more  closely,  it 
will  be  seen  that  there  is  a  vast  difference  between,  say, 
a  condition  caused  by  the  echinococcus  or  by  the  round 
worm,  in  which  the  effects  are  largely  mechanical  and  in 
which  relatively  little  poison  is  produced  by  the  parasite, 
and  the  disease  diphtheria  caused  by  the  diphtheria 
bacillus,  in  which  the  diphtheria  bacilli  have  little  or  no 
action  mechanically,  but  elaborate  virulent  chemical 
poisons  which  cause  a  general  intoxication.  Some  parasites 
also  may  produce  a  general  infection,  e.g.  anthrax,  others 
only  a  local  infection,  e.g.  ringworm. 

Parasites  may  therefore  be  divided  into  infective  and 
non-infective,  though  there  is  a  series  of  connecting  links 
between  these,  and  the  two  groups  cannot  be  sharply 
separated.  The  infective  parasites  are :  (1)  vegetable 
micro-organisms,  chiefly  bacteria,  a  few  yeasts  and  some 
moulds  ;  (2)  many  protozoa  ;  and  (3)  a  few  metazoa, 
generally  worms.  The  non-infective  parasites  are  the 
animal  parasites  generally,  particularly  many  worms. 

10 


146  A  MANUAL  OF  BACTERIOLOGY 

The  production  of  the  phenomena  of  disease  by  patho- 
genic organisms  has  been  ascribed  to  (1)  the  using  up  of 
the  oxygen  which  should  go  to  the  tissues  ;  (2)  the  using 
up  of  the  proteins  of  the  body  and  of  the  food  ;  (3)  the 
effects  of  plugging  of  the  vessels  by  the  microbes  ;  and 
(4)  the  effects  of  substances  or  "  toxins,"  having  a  poisonous 
action,  formed  by  the  microbes.  Of  these,  the  first  three 
are  quite  subsidiary,  embolism  and  thrombosis  being 
perhaps  the  most  important,  and  the  toxins  are  the  chief 
factors  which  induce  the  pathogenic  effects.  These  toxins 
are  substances  of  a  very  complex  composition,  probably 
allied  to  the  proteins  ;  in  some  instances  they  seem  to  be 
of  the  nature  of  enzymes  or  ferments,  and  they  are  direct 
products  of  the  bacterial  cells.  The  toxins  of  most 
pathogenic  organisms,  e.g.  typhoid,  cholera,  plague,  etc., 
are  more  or  less  integral  parts  of  the  bacterial  cells  ;  they 
are  "  endotoxins,"  and  are  not  excreted  to  any  extent 
into  the  surrounding  medium,  but  may  gain  access  to  it  by 
autolysis  of  some  of  the  organisms.  A  few  organisms, 
notably  Bacillus  diphtherice  and  Bacillus  tetani,  produce 
extra- cellular  toxins  which  are  found  in  the  culture  liquid. 
The  toxins  are  classified  by  Sidney  Martin,1  as  follows 
(see  also  p.  39)  : 

(1)  Poisons  produced  by  the  digestive  or  the  destructive 
action   of  bacteria   on  proteins  in  the  culture   medium. 
Examples  of  these  are  the  poisons  of  the  Bacillus  anthracis 
and  of  the  pus-producing  staphylococci. 

(2)  Poisons  which  are  the  result  of  the  digestive  or 
destructive  action  of  bacteria  on  proteins,  but  formed  as 
an  excretion  (the  toxin)  of  the  bacterium.     The  Bacillus 
diphtherice  is  the  best  example  of  this.     A  similar  com- 
bination of  poisons  is  found  in  snake- venom. 

(3)  Poisons  which  are  excretions  only,   such  as  those 
produced  by  the  tetanus  bacillus. 

1  Manual  of  General  Pathology,  p.  76. 


THE  INFECTIVE  PROCESS  147 

(4)  Poisons  which  are  typically  intra-cellular,  but  which 
may  also  be  excretory.  The  poisons  produced  by  the 
typhoid  bacillus,  the  Bacillus  coli,  the  Bacillus  enteritidis 
of  Gaertner,  and  the  cholera  vibrio  belong  to  this  group. 

Thiele  and  Embleton1  suggest  that  the  toxins  of  bacteria 
are  really  cleavage  products  derived  from  their  cellular 
proteins  under  the  influence  of  ferments  present  in  the 
body  of  the  host.  These  cleavage  products  are,  however, 
toxic  only  at  a  certain  stage  of  their  disintegration.  Given 
the  power  of  existing  and  multiplying  in  the  body  of  the 
host,  the  pathogenicity  of  a  bacterium  depends  on  the 
quantity  and  consequent  activity  of  the  ferments  of  the 
host.  A  certain  degree  of  ferment  activity  renders  the 
cleavage  products  of  the  bacterio-protein  toxic,  a  further 
degree  of  ferment  activity  carries  the  disintegration  so  far 
that  the  cleavage  products  are  no  longer  toxic.  A 
bacterium  may  therefore  be  harmless  to  a  host  if  the 
latter  (a)  has  no  ferments  capable  of  digesting  its  bacterio- 
protein  ;  (b)  has  such  a  poor  supply  of  ferments  that  the 
bacterio-protein  is  so  slowly  disintegrated  that  toxic 
products  never  attain  a  sufficient  concentration  to  be 
harmful ;  (c)  has  such  a  plentiful  supply  of  ferments  that 
the  cleavage  of  the  bacterio-protein  rapidly  passes  beyond 
the  toxic  stage.  A  harmless  bacterium,  e.g.  B.  megaterium> 
may  be  rendered  pathogenic  if  suitable  ferments  can  be 
produced  in  the  host  to  bring  about  the  necessary  dis- 
integration of  its  bacterio-protein. 

The  Infective  Process 

With   regard   to   the   pathogenic   micro-organisms,    or 

disease  germs,  Koch  laid  down  the  following  conditions, 

which   have   been    termed    "  Koch's   postulates,"    which 

must  be  complied  with  before  the  relation  of  an  organism 

1  Lancet,  vol.  i,  1913,  pp.  234  and  332. 


148  A  MANUAL  OF  BACTERIOLOGY 

to  a  disease  process  can  be  said  completely  to  be  demon- 
strated : 

(1)  The  organism  in  question  must  be  present  in  the 
tissues,  fluids,  or  organs  of  the  animal  affected  with,  or 
dead  from,  the  disease. 

(2)  The  organism  must  be  isolated  and  cultivated  out- 
side the  body  on  suitable  media  for  successive  generations. 

(3)  The  isolated  and  cultivated  organism,  on  inoculation 
into  a  suitable  animal,  should  reproduce  the  disease. 

(4)  In  the  inoculated  animal  the  same  organism  must 
be  found. 

To  these  may  be  added  : 

(5)  Chemical    products    with    a    similar    physiological 
action  may  be  obtainable  from  the  artificial  cultures  of  the 
micro-organism,  and  from  the  tissues  of  man  or  animals 
dead  of  the  disease. 

(6)  Specific  serum  and  other  reactions,   agglutinative, 
bacteriolytic,    complement    fixative,    etc.,    are    generally 
obtainable,  under  certain  conditions,  if  the  blood  of  the 
infected  person  or  animal  be  allowed  to  act  on  the  specific 
organism  producing  the  infection. 

It  is  true  that  one  or  more  of  these  conditions  may  not 
be  fulfilled  in  all  cases,  but  on  general  evidence  the  disease 
is  classed  as  infective. 

The  modes  of  infection,  or  entrance  of  the  infective 
agent  into  the  body,  are  varied.  The  infective  agent 
may  enter  by  (1)  the  gastro-intestinal  tract,  e.g.  typhoid, 
cholera,  and  glanders ;  (2)  the  respiratory  tract,  e.g. 
pneumonia  and  influenza,  and  occasionally  typhoid, 
plague,  etc.  ;  (3)  by  inoculation,  not  necessarily  only  of 
the  skin,  but  also  of  the  mucous  membranes,  e.g.  the 
septic  diseases,  glanders,  tetanus,  etc.  The  extreme  infec- 
tivity  of  some  diseases — e.g.  variola,  scarlatina,  influenza, 
etc. — may  be  due  to  the  fact  that  infection  takes  place 
by  the  respiratory  tract.  In  certain  instances  the 


ANTI-BODIES  149 

infection  is  conveyed  in  some  special  way,  e.g.  by 
mosquitoes  in  malaria  and  in  yellow  fever.  Nor  is 
infection  necessarily  confined  to  one  mode  of  entrance  ; 
in  plague,  for  example,  infection  by  the  skin  is  com- 
monest in  some  epidemics,  but  it  is  not  infrequent  by 
the  respiratory,  and  may  occur  by  the  digestive,  tract. 
The  infecting  agent  may  remain  localised,  giving  rise  to 
a  local  infection,  or  it  may  be  widespread  through  the 
body,  a  septiccemia1  or  general  infection.  The  absorption 
of  chemical  products  from  a  local  site  of  infection  may 
produce  general  symptoms  ;  this  is  intoxication,  as  occurs 
in  cholera,  in  which  the  microbe  is  limited  to  the  bowel, 
in  the  early  stage  of  diphtheria,  in  which  the  diphtheria 
bacillus  is  limited  to  the  membrane,  and  in  a  local  abscess. 
Fever  is  usually  one  of  the  results  both  of  intoxication 
and  of  general  infection. 

Infection,  if  recovery  ensues,  is  usually  followed  by 
remarkable  alterations  in  the  blood  and  tissues.  One 
of  these  is  the  production  of  immunity  or  insusceptibility 
to  the  same  infecting  agent ;  this  will  be  considered  later 
(p.  195).  Agglutinins,  substances  which  cause  clumping 
of  the  infecting  organism,  are  also  generally  produced 
(p.  185). 

Anti-Bodies 2 

Another  remarkable  property,  and  one  of  considerable 
importance  in  immunity,  conferred  by  the  injection  into 
an  animal  of  complex  substances,  such  as  bacterial  toxins, 
bacteria,  blood- corpuscles,  cells  and  cellular  proteins, 
ferments,  etc.,  is  the  development  of  anti-bodies.  Thus 

1  "  Septicaemia  "  and  "  a  septicaemia  "  have  different  meanings.     The 
former  is  applied  to  a  general    infection  with    the    so-called    septic 
organisms,  the  latter  to  a  general  infection  with  any  organism. 

2  All  the  subjects  dealt  with  in  the  subsequent  portion  of  this  chapter 
are   discussed  in   detail  by  Emery,  Immunity  and  Specific   Therapy, 
1909. 


150  A  MANUAL  OF  BACTERIOLOGY 

an  animal  injected  with  sub-lethal  doses  of  a  bacterial 
toxin,  e.g.  diphtheria  toxin,  acquires  a  tolerance  towards 
the  toxin,  becomes  immunised,  and  a  substance  is  de- 
veloped in  the  blood  that  antagonises  the  toxin  which 
was  injected  ;  this  substance  is  known  as  antitoxin.  If 
bacteria  be  injected,  the  fresh  blood  in  vitro  has  a  solvent 
action  on  the  bacteria  (bacteriolysis)  ;  if  blood- corpuscles 
be  injected,  the  fresh  blood  has  a  solvent  action  on  the 
same  kind  of  blood-corpuscles  (haemolysis) ;  if  cells  be 
injected,  the  blood  has  a  solvent  action  on  the  cells 
(cytolysis),  and  so  on.  If  ferments  be  injected,  anti- 
ferments  are  formed  and  will  prevent  the  specific  action 
of  the  ferment.  With  doubtful  exceptions,1  it  is  only 
complex  bodies  of  protein  nature,  or  allied  to  the  proteins, 
which  give  rise  to  the  production  of  anti-bodies  on  inocula- 
tion ;  alkaloids,  carbohydrates,  mineral  poisons,  etc.,  do 
not  give  rise  to  anti-bodies,  though  some  insusceptibility 
to  them  may  be  produced  (see  also  p.  206).  Any  substance 
which  gives  rise  to  an  anti-body  may  be  termed  an  anti- 
gen. These  anti-bodies,  etc.,  may  first  be  considered, 
after  which  immunity  will  be  discussed. 

Anti-bodies  are  probably  formed  for  the  most  part  in  the 
spleen,  lymph-glands  and  bone-marrow  by  leucocytes,  or 
by  endothelial  cells,  or  by  both. 

ANTITOXINS. — The  anti-bodies  produced  by  the  inocu- 
lation of  an  animal  with  bacterial  toxins  or  toxic  proteins 
(e.g.  ricin,  abrin,  and  snake-venom)  are  known  as  anti- 
toxins, and  are  of  considerable  practical  importance.  An 
animal  injected  with  increasing  amounts  of  the  toxin 
acquires  a  high  degree  of  immunity,  and  its  blood-serum 
injected  into  a  second  animal  confers  on  the  latter  a 
similar  immunity  against  the  same  toxin,  but  not  against 
other  toxins ;  the  serum  is  specific.  The  anti-serum 

1  Ford  has  described  the  formation  of  an  anti-body  by  the  injection 
of  a  poisonous  glucoside  derived  from  fungi. 


ANTITOXINS  151 

formed  by  the  injection  of  toxin  is  antitoxic  and  not 
anti-microbic,  and  the  diphtheria  bacillus  will  grow  and 
multiply  in  diphtheria  antitoxin.  Since,  however,  the 
pathogenic  effects  of  an  organism  such  as  the  diphtheria 
or  the  tetanus  bacillus  are  caused  by  the  toxin  which  it 
forms,  the  antitoxin  will  counteract  the  effects  of  the 
micro-organism  as  well  as  of  its  toxin.  The  neutralisa- 
tion of  the  micro-organism,  however,  may  not  be  quite 
complete,  a  certain  amount  of  local  reaction  or  necrosis 
ensuing. 

Antitoxins  are  prepared  by  injecting  animals — prefer- 
ably horses,  but  goats,  rabbits,  etc.,  may  also  be  employed 
• — with  bacterial  toxins  or  with  cultures. 

With  those  organisms  which  produce  potent  toxins 
such  as  diphtheria  and  tetanus,  it  is  customary  to  grow 
the  organism  in  a  fluid  medium  so  that  an  active  and 
virulent  toxin  is  obtained.  The  culture  is  then  filtered 
through  a  Berkefeld  or  Pasteur-Chamberland  filter  and  the 
toxic  filtrate  inoculated  subcutaneously  into  an  animal, 
generally  a  horse,  commencing  with  sub- lethal  doses. 

The  dose  of  toxin  can  be  gradually  increased,  and  con- 
currently with  the  increase  in  insusceptibility  the  blood- 
serum  acquires  antitoxic  properties.  The  treatment  is 
tedious,  and  the  activity  of  the  antitoxic  serum  is  largely 
dependent  upon  the  amount  and  activity  of  the  toxin 
injected.  The  requisite  degree  of  strength  having  been 
attained,  the  horse  is  bled  with  aseptic  precautions,  the 
blood  is  allowed  to  coagulate,  and  the  serum  is  bottled 
for  use.  Antitoxin  may  be  obtained  in  a  concentrated 
form  by  "  salting  out "  the  globulin  constituents  of  an 
antitoxic  serum  (p.  167),  and  a  dried  product  may  be 
prepared  by  evaporating  the  serum  to  dryness  in  vacuo 
at  40°  C.  ( 10  c.c.  serum  =  1  grm.  dry  residue). 

The  mode  of  production  of  the  antitoxin  by  the  injection 
of  the  toxin  has  been  the  subject  of  various  theories.  By 


152  A  MANUAL  OF  BACTERIOLOGY 

some  it  has  been  supposed  that  the  antitoxin  is  modified 
toxin,  the  modification  being  brought  about  by  the  vital 
activities  of  the  cells.  But  the  amount  of  antitoxin 
produced  does  not  necessarily  bear  any  relation  to  the 
quantity  of  toxin  injected.  Woodhead  records  instances 
in  which  the  amount  of  antitoxin  formed  amounted  to 
40,000  times  the  equivalent  amount  of  toxin  injected, 
bleeding  the  animal  only  temporarily  reduces  the  anti- 
toxin content  of  the  serum,  and  substances  which  increase 
the  secretive  properties  of  glandular  cells,  such  as  pilo- 
carpine,  enormously  increase  the  output,  so  to  speak,  of 
antitoxin. 

In  view  of  these  facts  Ehrlich  elaborated  his  "  side- 
chain  theory,"  a  theory  which,  whether  it  be  the  real 
explanation  or  no,  has  received  a  considerable  amount  of 
experimental  support,  and  has  had  far-reaching  effects 
in  stimulating  research.  Ehrlich  believes  that  the  chemical 
activities  which  are  the  manifestations  of  the  vital 
activities  of  the  living  cell  are  due  to  a  very  large  nucleus 
or  chemical  molecule  having  a  ring  structure,  analogous 
to  the  benzene  ring,  and  having  attached  to  it  a  number 
of  atomic  groups  or  "  side- chains."  A  "  side- chain  "  is 
an  atomic  group,  a  carbon  atom  of  which  is  linked  to  one 
of  the  carbon  atoms  in  a  ring.  These  atomic  groups  or 
side-chains  are  unstable  in  nature,  and  enter  freely  into 
combination  with  other  suitable  groups  should  these  be 
presented  to  them,  and  thus  the  physiological  activities 
of  the  cell,  assimilation,  nutrition,  etc.,  are  carried  out 
(Fig.  26).  Now  Ehrlich  supposes  that  antitoxin  is  merely 
an  excess  of  certain  side-chains  which  are  normally  present 
and  subserve  some  of  the  ordinary  functions  of  the  cell 
and  which  have  become  free  in  the  blood.  The  antitoxins 
being  specific,  by  this  assumption  the  difficulty  is  obviated 
of  supposing  that  special  chemical  groups  or  molecules 
exist  preformed  ready  to  combine  with  a  number  of 


SIDE-CHAIN  THEORY 


153 


different  toxins  on  the  remote  chance  that  some  one  of  these 
may  at  some  time  or  other  come  within  the  particular 
sphere  of  action  of  one  of  those  groups.  Moreover,  small 
amounts  of  anti-bodies,  such  as  antitoxin,  bacteriolysin, 
agglutinin,  etc.,  are  met  with  in  normal  untreated  animals 
and  in  man.  While  some  have  supposed  that  the  small 
amount  of  diphtheria  antitoxin  (equivalent  to  half  a  unit 


FIG.  26. — Diagram  to  represent 
the  cell  with  its  various  com- 
bining groups  or  side-chains. 
(After  Ehrlich.) 


FIG.  27. — First  stage  in  anti- 
toxin formation.  (Black  = 
toxin  molecule.  (After  Ehr- 
lich.) 


or  so)  present  in  human  blood-serum  is  due  to  an  infection 
with  the  diphtheria  bacillus  (not  necessarily  an  attack  of 
diphtheria),  it  seems  more  rational  to  suppose  that  this 
antitoxin  is  due  to  a  natural  liberation  of  such  side- chains 
from  the  protoplasm  and  that  artificial  antitoxin  pro- 
duction is  merely  a  very  great  stimulation  of  this  natural 
process. 

The  toxin  molecule,  according  to  Ehrlich,  possesses  at 
least  two  fixative  atomic  groups  or  side-chains.  One 
of  these,  the  "  haptophore  group,"  conditions  the  union 
of  the  toxin  molecule  with  cell-protoplasm  ;  the  other, 
the  "  toxophore  group,"  conditions  its  toxic  action. 
Similarly,  in  order  that  the  cell  may  suffer  the  full  effect 
of  the  action  of  the  toxin,  it  also  must  possess  two  receptive 


154  A  MANUAL  OF  BACTERIOLOGY 

groups  or  side-chains  having  a  maximum  affinity  for  the 
haptophore  and  toxophore  groups  of  the  toxin  ;  these 
may  be  termed  the  "  receptor  "  and  "  toxophile  "  groups 
respectively  (see  Fig.  31).  The  relationship  of  each 
fixative  group  of  the  corresponding  groups — viz.  that  of 
the  toxin  and  that  of  the  side-chain  of  the  cell— must  be 
most  intimate,  and  analogous  to  the  relations  to  each 


FIG.  28. — Second  stage  in  anti-  FIG.  29. — Third  stage  in  anti- 
toxin formation.  (After  Ehr-  toxin  formation.  Side-chains 
lich.)  beginning  to  be  produced  in 

excess.     (After  Ehrlich.) 

other  of  a  male  and  a  female  screw  (Pasteur)  or  of  a  lock 
and  its  key  (E.  Fisher). 

The  genesis  of  antitoxin  on  the  "  side- chain  theory  " 
takes  place  in  the  following  manner  :  Toxin  being  intro- 
duced, the  haptophore  groups  of  the  toxin  molecules  unite 
with  the  particular  receptor  side-chains  of  the  proto- 
plasm for  which  they  have  an  affinity  (Fig.  27).  By  this 
combination  the  physiological  activities  of  the  cell  are 
interfered  with,  a  defect  is  created,  the  cell  is  damaged 
(it  is  only  necessary  to  consider  the  case  of  one  cell,  or, 
more  strictly  of  one  molecular  group  of  the  cell-protoplasm). 
But  through  its  recuperative  powers  the  cell  soon  recovers 
by  the  formation  of  new  receptor  side- chains  to  take  the 
place  of  those  which  have  been  put  out  of  action.  On 
injecting  more  toxin,  this  combines  with  these  new  receptors 


RECEPTORS 


155 


arid  a  defect  is  again  created  (Fig.  28).  Once  more  the 
cell  responds,  and  a  fresh  series  of  receptors  is  developed 
(Fig.  29).  But  by  this  continual  stimulation,  as  it  were, 
the  cell  commences  to  form  the  particular  receptors  in 
excess  of  that  needed  to  repair  the  defect  created,  and  ultimately 
these  receptors  are  reproduced  in  such  numbers  that 
they  no  longer  all  remain  attached  to  the  cell  but  some  be- 
come free  in  the  plasma  (Fig.  30). 
These  receptor  sidechains,  detached 
from  the  cell  and  floating  free  in 
the  blood-stream,  constitute  the  anti- 
toxin. This  excessive  production 
of  side-chains  after  stimulation  by 
repeated  injections  of  toxin  is  not 
a  phenomenon  confined  to  anti- 
toxin formation,  but  is  a  general 
physiological  law  enunciated  by 
Weigert ;  as  a  result  of  repeated 
stimulation,  over-production  or 
hyper-compensation  is  the  rule 
and  is  met  with  in  various 

pathological  processes.  Ehrlich  has  termed  the  diverse 
free  receptors  which  occur  in  the  body  fluids  in  various 
circumstances  "  haptines." 

The  existence  of  both  haptophore  and  toxophore  groups 
in  the  toxin  molecule  is  suggested  by  the  following  experi- 
ments. Tetanus  toxin  injected  into  the  blood-stream  of 
an  animal  rapidly  disappears,  within  a  few  seconds  of 
the  injection,  and  even  if  the  animal  be  at  once  bled, 
the  blood  withdrawn  being  replaced  by  fresh  blood, 
tetanus  ensues,  but  not  until  after  the  lapse  of  an  in- 
cubation period  of  some  hours.  The  tetanus  toxin, 
therefore,  immediately  becomes  fixed  or  anchored  to  the 
tissues  of  the  central  nervous  system.  Evidently  the 
toxin  molecules  enter  at  once  into  combination  with  the 


FIG.  30. — Fourth  stage  in 
antitoxin  formation. 
Side-chain,  i.e.  antitoxin, 
free  in  the  blood.  (After 
Ehrlich.) 


156  A  MANUAL  OF  BACTERIOLOGY 

nerve-tissues  by  means  of  their  haptophore  groups  ;  this 
after  a  time  brings  the  cells  within  the  sphere  of  influence 
of  the  toxophore  groups,  and  after  a  certain  incubation 
period  toxic  symptoms  ensue.  The  affinity  of  tetanus 
toxin  for  nerve  tissues  may  be  shown  in  another  way.  If 
fresh  guinea-pig  brain  be  emulsified  with  tetanus  toxin, 
the  emulsion  will  be  found  to  be  innocuous  on  injection, 


FIG.  31. — Diagrammatic  scheme  to  represent  the  union  of  toxin 
(black)  with  the  cell.  In  A  the  toxin  is  attached  to  the  pro- 
toplasm by  the  union  of  the  haptophore  and  receptor  groups. 
In  B  the  toxophore  and  toxophile  groups  have  also  united, 
and  poisoning  now  ensues. 

owing  to  a  combination  between  the  two  having  taken 
place.  The  cerebral  cortex  of  a  highly  susceptible  animal 
(e.g.  mouse)  has  a  marked  neutralising  power,  of  a  less 
susceptible  animal  (e.g.  rabbit,  fowl)  a  feebler,  and  of  an 
insusceptible  animal  (e.g.  frog,  tortoise)  no  neutralising 
power.1  Moreover,  both  diphtheria  and  tetanus  toxins 
may  be  converted  into  non- toxic  modifications  ("  toxoids  ") 
which  to  some  extent  retain  the  power  of  immunising 
and  of  producing  antitoxin  on  inoculation,  and  of  com- 
bining with  antitoxin  :  that  is  to  say,  according  to  Ehrlich, 

1  The  combination  of  brain  matter  with  tetanus  toxin  seems  to  be 
specific  and  of  the  same  order  as  that  between  antitoxin  and  toxin. 
See  Noon,  Journ.  of  Hyg.,  vol.  vii,  1907,  p.  101,  and  Besredka  and 
Bordet,  Ann.  de  Vlnst.  Past.,  xvii,  1903. 


NEUTRALISATION  OF  TOXIN  157 

the  toxophore  groups  have  been  destroyed  while  the 
haptophore  groups  remain  unaffected.  It  is  the  presence 
of  the  haptophore  group  which  conditions  the  union  of 
toxin  with  antitoxin.  Thus,  if  toxin  be  injected  into 
blood  containing  antitoxin,  the  haptophore  groups  of 
the  toxin  unite  with  the  free  receptor  groups,  i.e.  with 
the  antitoxin  (Fig.  32),  and  therefore  the  toxophore  groups 
cannot  exert  their  influence 
because  the  toxin  is  now 
unable  to  unite  with  the  pro- 
toplasm, its  haptophore  or 
binding  groups  being  already 
occupied. 

In  a  poisonous  toxin,  such 
as  diphtheria  or  tetanus  toxin, 
the  toxophore  group  is  more 
readily  destroyed  than  the  FIG.  32.— Neutralisation  of  toxin 
haptophore  group,  and  by  ^~h,in  the  "^ 
heating  a  toxin  for  some 

time  to  60°-70°  C.  its  toxicity  is  destroyed,  but  it  still 
retains  an  affinity  for  antitoxin.  If  some  antitoxin  be 
mixed  with  such  heated  toxin  it  will  be  found  that  the 
capacity  of  the  former  for  neutralising  active  toxin  is 
much  diminished — in  other  words,  although  the  toxophore 
groups  of  the  heated  toxin  have  been  destroyed,  the 
binding  or  haptophore  groups  still  remain.  Toxin  which 
has  been  kept  for  some  time  decreases  in  toxicity,  but 
retains  the  power  of  combining  with  antitoxin,  again 
showing  that  haptophore  or  binding  groups  are  present 
(such  derivatives  of  toxin  possessing  haptophore  groups 
are  termed  "  toxoids  ").  Wassermann  and  Bruck  have 
obtained  presumptive  evidence  of  the  existence  of  the 
second  stage  in  antitoxin  formation,  viz.  the  increased 
production  of  receptors  by  the  cells.  Using  tetanus  toxin 
which  had  been  kept  for  some  time  and  had  lost  its 


158  A  MANUAL  OF  BACTERIOLOGY 

toxicity,  but  which  still  combined  with  antitoxin — that 
is,  toxoids  with  haptophore  groups  still  present — they 
found  that  on  injecting  it  into  animals  no  antitoxin 
was  formed  as  a  result  of  the  injection.  They  then  per- 
formed some  experiments  based  on  the  following  line  of 
reasoning  :  If  the  old  non-poisonous  tetanus  toxin  con- 
taining these  toxoids  be  first  injected  into  an  animal,  and 
after  a  short  interval,  some  fresh,  actively  poisonous 
tetanus  toxin,  more  of  the  active  toxin  ought  to  be  required 
to  kill  this  animal  than  a  normal  one,  because,  owing  to 
the  previous  toxoid  injection,  part  of  the  cell  receptors 
susceptible  to  tetanus  toxin  are  already  occupied.  Pro- 
vided Ehrlich's  theory  be  correct,  so  that  this  binding 
of  the  toxoid  really  occurs,  the  conditions  should  be  entirely 
different,  when,  instead  of  injecting  the  toxin  shortly 
after  the  toxoid,  a  longer  time  elapsed — one  to  three 
days — before  the  injection  of  the  active  tetanus  toxin. 
For  in  that  case  Weigert's  law  should  come  into  play 
and  the  receptors  should  have  increased  in  number — i.e. 
the  organism  would  now  possess  more  sensitive  groups 
than  before.  This  should  be  manifest  by  the  fact  that,  in 
contrast  to  the  first  experiment,  the  fatal  dose  of  active 
tetanus  toxin  ought  now  to  be  smaller  than  previously ; 
in  other  words,  a  smaller  dose  should  now  tetanise  the 
animal  in  a  shorter  time.  The  experiments  yielded  results 
which  were  exactly  in  accordance  with  these  theoretical 
considerations.  A  guinea-pig  was  injected  with  some  of 
the  non-poisonous  toxoid,  and  then,  one  hour  later^  with 
the  active  tetanus  toxin.  It  was  found  that  much  more 
toxin  was  required  to  kill  this  animal  than  a  normal  guinea- 
pig  of  equal  size.  If,  on  the  contrary,  an  interval  of  one 
to  three  days  were  allowed  to  elapse,  it  was  then  found 
that  a  dose  of  tetanus  toxin  which  would  not  even  tetanise 
a  normal  guinea-pig  was  sufficient  to  kill  this  one. 
The  fact  that  no  antitoxin  is  formed — i.e.  no  receptors 


ABSORPTION  OF  TOXIN  159 

are  thrust  off — by  the  single  injection  of  the  non- 
poisonous  toxin,  or  toxoid,  Wassermann  ascribes  to  the 
lack  of  stimulus  which  he  suggests  resides  in  the  toxo- 
phore  groups. 

The  slow  combination  of  the  haptophore  and  receptor 
groups  has  been  proved  by  Wassermann  in  another  way. 
The  researches  of  Meyer  and  Ransom  have  shown  that 
tetanus  toxin  is  absorbed  by  the  nerve- trunks,  not  by 
the  blood  and  lymph-channels,  while  tetanus  antitoxin 
is  absorbed  by  the  latter — the  blood  and  lymph- channels. 
Adrenalin  is  a  substance  which  strongly  contracts  the 
capillaries,  and  thus  tends  to  block  absorption  in  a  parti- 
cular area.  The  following  experiment  was  devised : 
Tetanus  toxin  and  antitoxin  were  mixed  in  such  propor- 
tions that  the  mixture  was  innocuous  to  animals,  i.e.  it 
was  just  neutral.  If  this  mixture  be  injected  into  the 
hind  paw  of  a  guinea-pig  no  tetanus  develops.  When, 
however,  some  adrenalin  is  injected  into  the  hind  paw  of 
a  similar-sized  guinea-pig,  and  a  few  minutes  are  allowed 
to  elapse  so  that  the  capillaries  may  contract,  and  then  the 
mixture  of  toxin  and  antitoxin  is  injected,  typical  tetanus 
ensues.  The  explanation  of  this  is  that  the  channel  of 
absorption  for  the  tetanus  antitoxin,  the  vessels,  is  blocked 
by  the  adrenalin,  while  that  for  the  toxin,  the  nerve  path, 
remains  open.  The  toxin  and  antitoxin  had  not  yet 
combined,  or  such  combination  as  had  occurred  is  a  loose 
one  and  becomes  dissociated,  and,  therefore,  the  toxin 
travelled  along  the  nerves  to  the  central  nervous  system 
with  the  production  of  tetanus. 

The  experiment,  however,  succeeds  only  within  a  certain 
period,  not  exceeding  an  hour  after  mixture  of  the  toxin 
and  antitoxin,  because  after  this  the  toxin-antitoxin 
combination  becomes  a  stable  one. 

If  a  longer  time — say  three  or  four  hours — is  allowed 
to  elapse,  it  will  be  found  that,  even  in  the  adrenalin 


160  A  MANUAL  OF  BACTERIOLOGY 

animal,  no  tetanus  is  produced,  because  by  this  time  the 
combination,  previously  a  loose  one,  has  become  so  stable 
that  the  substances  can  no  longer  be  dissociated.  This 
union  can  be  hastened  by  employing  more  tetanus  anti- 
toxin, for  with  an  excess  of  antitoxin,  even  after  only  half 
an  hour,  it  is  impossible  by  means  of  adrenalin  to  free 
the  tetanus  toxin.  This  experiment,  therefore,  shows  that 
the  combination  of  tetanus  toxin  with  antitoxin  takes 
place  slowly  and  is  at  first  a  loose  one,  and  that  the  union 
becomes  firmer  and  firmer  with  lapse  of  time.  It  also 
suggests  the  possibility  of  hastening  the  combination  by 
increasing  the  amount  of  antitoxin — a  point  of  consider- 
able practical  value  in  serum  therapy. 

The  above  considerations  are  of  importance  in  the  antitoxin 
treatment  of  disease.  Antitoxin,  in  the  strict  sense,  is  not  anti- 
microbic,  and  therefore  antiseptic  treatment  of  the  throat  in 
diphtheria,  and  of  the  wound  in  tetanus,  should  be  pursued.  The 
fact  that  the  toxophore  group  of  the  toxin  does  not  come  into 
action  as  a  rule  for  many  hours  at  least  (an  exception  is  snake - 
venom)  is  a  fortunate  coincidence,  for  the  antitoxin  may,  there- 
fore, act  before  tissue  damage  has  occurred.  Antitoxin  cannot 
repair  tissue  damage  if  this  has  been  produced  by  the  toxin,  but 
it  can,  and  does,  prevent  the  occurrence  of  further  damage  by 
neutralising  any  fresh  amounts  of  toxin  that  may  be  absorbed. 
Hence  the  necessity  for  early  treatment.  Toxin  already  anchored 
to  the  tissues  by  its  haptophore  group  may  for  some  time  be  dis- 
sociated from  them  if  a  multiple  of  the  simple  neutralising  dose  of 
antitoxin  be  injected,  and  the  quantity  necessary  to  accomplish 
this  rises  rapidly  as  the  interval  between  the  introduction  of  the 
toxin  and  of  the  antitoxin  increases  ;  hence  the  necessity  for  the 
use  of  antitoxin  in  large  excess.  Probably  the  union  between  tissue 
and  toxin  at  first  is  a  loose  one,  and  a  large  amount  of  antitoxin 
by  mass  action  transfers  the  affinity  of  the  toxin  from  the  tissues 
to  itself.  It  must  be  clearly  recognised  that  colloidal  reactions 
(to  which  category  that  between  antitoxin  and  toxin,  anti-body 
and  antigen,  belongs)  differ  considerably  from  ordinary  chemical 
reactions. 

An  essential  condition  in  antitoxic  treatment  is  the  administration 
of  a  sufficient  amount  of  anti-serum,  and  this  does  not  depend  on 


NEUTRALISATION  OF  TOXIN  161 

the  actual  volume  of  serum  injected.  The  anti-serum  may  be 
regarded  as  a  solution  containing  a  variable  amount  of  the  anti- 
toxic or  anti-microbic  constituent,  and  for  therapeutic  use  its 
strength  must  be  ascertained,  and  is  for  convenience  described  in 
arbitrary  unite. 

The  dose  of  antitoxin  is  dependent  upon  the  gravity  of  the 
disease,  and  not  on  the  age  of  the  patient,  for  evidently  just  as 
much  toxin  may  be  formed  in  a  child  as  in  an  adult.  The  anti- 
toxins are  strictly  specific  ;  diphtheria  antitoxin,  for  example,  has 
not  the  slightest  influence  in  tetanus. 

To  obtain  an  immediate  reaction  to  antitoxin  it  should  be 
administered  intra-venously.  A  subcutaneous  injection  may  not 
be  completely  absorbed  in  less  than  thirty-six  hours,  an  intra- 
muscular injection  is  much  more  rapidly  absorbed. 

In  cases  of  mixed  infection,  e.g.  where  diphtheria  bacilli  are 
associated  with  streptococci  or  staphylococci,  the  diphtheria  anti- 
toxin will  have  no  influence  on  the  streptococcic  or  staphylococcic 
infection. 

The  complications  and  accidents  of  antitoxin  treatment  are  few 
and  usually  unimportant.  Abscess  and  other  local  troubles  at  the 
seat  of  inoculation  should  not  occur  if  proper  antiseptic  precautions 
be  taken.  Urticaria  or  other  rashes  and  joint  pains  are  by  far  the 
most  troublesome  complications.  These  are  due  to  the  injection 
of  foreign  serum,  and  not  to  the  antitoxin,  for  the  serum  of  an 
untreated  horse  produces  a  like  effect.  Repeated  injections  of 
serum  at  short  intervals  may  be  continued  for  a  long  period  without 
inducing  more  disturbance  than  that  caused  by  one  or  two  or  a  few 
injections,  but  if  twelve  days  or  more  elapse  between  two  injections 
a  condition  of  "  supersensitation,"  due  to  anaphylaxis,  ensues 
(see  p.  168).  This  consists  in  the  rapid  appearance  of  rashes,  joint 
pains,  pyrexia,  etc.,  or  even  of  grave  symptoms,  faintness,  vomiting, 
dyspnoea,  convulsions,  collapse,  etc. 

Anti-sera  may  be  used  as  prophylactics,  but  the  immunity 
produced  by  them  does  not  last  more  than  three  weeks. 

Various  hypotheses  have  been  advanced  to  explain 
the  manner  in  which  toxin  is  neutralised  by  antitoxin. 
Roux  and  Buchner  suggested  that  the  antitoxin  in 
some  way  renders  the  cells  and  tissues  insusceptible 
to  the  toxin,  and  Buchner  performed  experiments  show- 
ing that  while  mice  are  more  susceptible  than  guinea- 
pigs  to  tetanus  toxin,  a  tetanus  toxin-antitoxin  mixture 

IT 


162  A  MANUAL  OF  BACTERIOLOGY 

which  is  just  neutral  for  mice  is  distinctly  toxic  for  guinea- 
pigs. 

To  explain  this  Ehrlich  suggested  that  there  may  be 
present  in  a  toxin  solution  several  toxic  substances,  some 
of  which  exert  a  toxic  action  on  the  guinea-pig  but  not 
on  the  mouse.  Madsen  and  Dreyer  showed  that  a  mixture 
of  diphtheria  toxin  and  antitoxin  which  is  innocuous  to 
guinea-pigs  on  subcutaneous  inoculation  is  lethal  to  rabbits 
on  intra- venous  injection,  and  in  order  to  explain  this 
Ehrlich  made  a  similar  assumption.  Morgenroth,  how- 
ever, found  that  the  difference  in  the  latter  case  depends 
on  the  mode  of  injection.  The  reaction  between  the 
toxin  and  antitoxin  takes  time  to  complete  :  there  is  an 
interval  probably  of  some  hours  at  20°  C.  before  equilibrium 
is  reached  (see  also  p.  163).  When  a  recently  prepared 
mixture  of  toxin  and  antitoxin  is  injected  subcutaneously, 
absorption  is  slow,  and  in  the  meanwhile  the  toxin  and 
antitoxin  combine,  but  when  the  mixture  is  injected  into 
the  veins,  the  toxin  is  fixed  by  the  tissues  before  it  has 
had  time  to  combine  with  the  antitoxin,  and  poisoning 
ensues.  If  the  mixture  be  kept  for  some  hours  before 
injection,  intravenous  injection  is  then  innocuous. 

Ehrlich  concluded  that  diphtheria  toxin  is  neutralised 
by  diphtheria  antitoxin  much  in  the  same  way  as  a  strong 
base  is  neutralised  by  a  strong  acid,  and  that  the  course  of 
neutralisation  suggests  the  presence  in  the  toxin  of  several 
toxic  and  atoxic  substances  (toxoids  and  toxones),  all 
of  which  combine  with,  though  they  have  different  affinities 
for,  the  antitoxin. 

Arrhenius  and  Madsen,  however,  believe  that  the  toxin- 
antitoxin  reaction  is  analogous  to  the  action  of  an  acid 
on  an  alcohol,  and  that  the  chemical  laws  of  mass  action 
apply  equally  to  the  two.  The  chief  reaction  is  considered 
to  be  between  two  substances  only,  toxin  and  antitoxin, 
that  it  is  reversible,  and  that  when  the  system  has  reached 


THE  TOXONE  EFFECT  163 

equilibrium,  a  fraction  of  toxin  and  also  of  antitoxin 
remains  free,  this  fraction  of  toxin  producing  the  "  toxone 
effect  "  (see  p.  165).  If  equivalent  quantities  of  acetic 
acid  and  alcohol  are  mixed,  the  reaction  is  never  complete  ; 
the  acid  and  alcohol  never  entirely  disappear,  because  the 
water  formed  reacts  with  the  ethyl  acetate,  re-converting 
it  into  acid  and  alcohol.  Such  a  reaction  is  termed  rever- 
sible, and  this  particular  case  could  be  thus  represented  : 


CH.COOH  +  CH.OHCH.COOCH       H0. 


Bordet  has  suggested  that  the  fixation  of  toxin  by 
antitoxin  is  an  adsorption  phenomenon,  similar  to  the 
fixation  of  a  dye  by  a  tissue. 

These  hypotheses  may  now  be  examined  more  in  detail. 
Ehrlich's  experiments  l  on  diphtheria  toxin  seemed  to 
show  that  the  neutralisation  of  toxin  by  antitoxin  follows 
the  laws  of  simple  chemical  combinations,  such  as  the 
neutralisation  of  a  strong  base  (NaOH)  by  a  strong  acid 
(HC1).  If  so,  it  would  be  expected  that  antitoxin  would 
neutralise  proportionate  amounts  of  toxin  ;  but  this  is 
not  so,  and  Ehrlich  was  forced  to  the  conclusion  that  toxin 
is  a  complex  mixture  of  proto-,  deutero-,  and  trito-  toxin, 
and  toxone,  with  different  toxicities  and  different  avidities 
for  antitoxin.  Moreover,  when  toxin  is  kept  it  decreases 
in  toxicity,  though  still  retaining  much  of  its  avidity  for 
antitoxin.  Ehrlich  assumed,  therefore,  that  the  toxin 
becomes  transformed  into  substances  termed  toxoids, 
which  are  non-toxic  but  retain  their  affinity  for  antitoxin 
(see  also  section  on  the  standardisation  of  diphtheria  anti- 
toxin). This  he  explained  as  due  to  destruction  of  the 
unstable  toxophore  groups,  with  the  retention  of  the 
more  stable  haptophore  groups.  That  the  neutralisation 
of  toxin  by  antitoxin  is  due  to  some  sort  of  union  between 

1  l-'w  Trans.  Jcnncr  Inst.  Prcv.  Mcd.,  vol.  ii,  p.  1  ;  Croonian  Led., 
Roy.  tioc.  Lond.,  1900  ;  and  p.  293. 


164  A  MANUAL  OF  BACTERIOLOGY 

the  two,  though  not  necessarily  chemical  combination  in 
the  strict  sense,  seems  to  be  proved  by  the  work  of  Martin 
and  Cherry.  Brodie,1  and  Martin  and  Cherry,2  making 
use  of  a  Chamberland  filter,  the  pores  of  which  had  been 
rendered  very  fine  by  saturating  with  gelatin,  found  that 
toxin  would  pass  through  such  a  filter  but  that  antitoxin 
would  not,  presumably  because  the  molecule  of  the  latter 
is  larger.  By  mixing  diphtheria  toxin  and  antitoxin  in 
such  proportion  that  the  latter  was  in  sufficient  quantity 
to  neutralise  the  toxin,  and  subjecting  the  mixture  to 
filtration  through  a  gelatin  filter,  the  filtrate  was  found 
to  be  non-toxic.  Now  since  toxin  can  pass  through  such 
a  filter,  the  inference  is  that  the  toxin  has  united  with 
the  antitoxin.  Using  snake- venom  and  its  anti-serum  or 
anti-venin,  another  method  was  employed.  The  anti- 
venin  is  destroyed  by  heating  to  68°  C.  for  ten  minutes, 
while  the  toxic  properties  of  the  venom  are  unaltered  by 
this  treatment.  By  making  mixtures  of  venom  and  anti- 
venin,  and,  after  a  certain  time  has  elapsed  for  the  inter- 
action to  take  place,  heating  to  68°  C.  for  ten  minutes, 
it  was  found  that  the  mixture  is  non-toxic,  pointing  to  the 
union  of  the  toxin  (venom)  with  the  antitoxin  (anti-venin). 
Calmette  had  performed  the  same  experiment  but  with 
a  different  result,  finding  his  mixtures  still  toxic  after 
heating.  Calmette,  however,  treated  his  solutions  almost 
immediately  after  mixing,  and  Martin  and  Cherry  point 
out  that  a  certain  time  must  be  allowed  to  elapse  for  the 
interaction  to  take  place,  and  noted  that  moderate  warming 
hastens  it,  as  is  the  case  with  all  chemical  interactions. 
For  instance,  they  found  that  one  mixture  of  venom  and 
anti-venin  allowed  to  interact  for  two  minutes,  five  minutes, 
and  ten  minutes  before  heating,  killed  the  animals  in 
thirteen  hours,  fifteen  hours,  and  twenty- three  hours 

1  Journ.  of  Path,  and  Bact.,  1897,  p.  460. 

2  Proc.  Roy.  Soc.  Lond.,  vol.  Ixiii,  1898,  p.  420. 


TOXIN- ANTITOXIN  REACTION  165 

respectively  (the  control  animal  with  the  same  dose  of 
venom  died  in  nine  hours),  but  after  fifteen  minutes  the 
same  mixture  rendered  the  animal  ill  but  it  survived,  while 
after  thirty  minutes  no  toxic  symptoms  ensued. 

At  one  time  it  was  stated  that  by  electrolysis  of  toxin 
small  amounts  of  antitoxin  are  formed,  but  this  is  very 
questionable.  Electrolysis  destroys  the  toxicity  of  toxins 
by  the  production  of  acids,  chlorine,  and  hypochlorites. 

Ehrlich's  views  have  been  opposed,  principally  on  physico- 
chemical  grounds.  Thus,  Danysz,  observed  that  if  ricin  or  diph- 
theria toxin  be  brought  into  contact  with  its  corresponding  anti- 
body, the  degree  of  neutralisation  depends  on  the  manner  of 
mixture.  If  the  toxin  be  added  to  the  antitoxin  in  two  fractions, 
allowing  a  considerable  time  to  elapse  between  the  additions,  the 
mixture  contains  a  much  larger  amount  of  free  toxin  than  is  the 
case  when  the  whole  (and  same)  amount  of  toxin  is  added  at  once 
to  the  antitoxin.  This  phenomenon,  known  as  the  "  Danysz  or 
toxone  effect,"  seems  inexplicable  if  toxin  and  antitoxin  have 
relations  the  same  as  a  strong  base  and  a  strong  acid. 

Arrhenius,  Dreyer,  and  Madsen  maintain  that  the  phenomena 
observed  in  the  toxin-antitoxin  reaction  are  explicable  on  the 
hypothesis  that  the  rate  of  reaction — avidity — of  the  toxin  decreases 
as  antitoxin  is  added,  that  the  interaction  is  a  slow  one,  and  that 
different  fractions  of  the  toxin  are  progressively  neutralised  by  the 
added  antitoxin,  but  more  and  more  slowly.  On  these  grounds 
they  consider  that  there  is  no  reason  to  regard  the  diphtheria  poison 
as  a  highly  complicated  body.  Whereas  Ehrlich  considers  the  toxin 
and  antitoxin  to  combine  with  great  avidity,  analogous  to  the 
combination  of  a  strong  base  with  a  strong  acid,  e.g.  NaOH  with 
HC1,  these  critics  believe  the  avidity  of  antitoxin  for  toxin  to  be 
feeble,  analogous  to  the  combination  of  ammonia  with  boric  acid, 
in  which  as  more  and  more  acid  is  added,  the  amount  of  free 
ammonia  decreases,  but  more  and  more  slowly,  in  correspondence 
with  a  hyperbolic  curve.  The  phenomena  can  be  calculated  accord- 
ing to  the  law  of  "  mass  action,"  there  being  an  equilibrium  between 

Free  NH3.   Free  H3O3B  =  y(NH4H2O3B)2 

vol.  vol.  vol. 

where  K  is  the  constant  of  dissociation.  The  curve  of  the  neutralisa- 
tion of  tetanolysin  by  anti-tetanolysin  corresponds  almost  exactly 
to  the  ammonia-boric-acid  curve. 


166  A  MANUAL  OF  BACTERIOLOGY 

Whereas  on  Ehrlich's  views  the  combination  of  toxin  and  anti- 
toxin would  be  represented  by  a  straight  line,  and  the  crude  toxin 
seems  to  be  composed  of  a  whole  series  of  different  toxins  and  sub- 
stances having  an  avidity  for  antitoxin,  on  this  hypothesis,  although 
the  greater  part  of  the  toxicity  of  toxin  is  removed  by  the  anti- 
toxin, the  latter  must  be  added  in  large  excess  before  the  toxicity 
completely  disappears,  and  the  course  of  neutralisation  would  be 
represented  by  a  hyperbolic  curve.  In  fact,  as  the  antitoxin  is 
added,  the  amount  of  free  toxin  diminishes  but  never  completely 
disappears.  There  comes  a  point,  of  course,  when  the  amount  of 
free  toxin  is  so  small  as  to  be  negligible  and  cannot  be  recognised 
by  the  ordinary  indicators  (blood-corpuscles,  animal  tests,  etc.). 
This  hypothesis  would  explain  the  fact  that  while  a  certain  amount, 
V,  of  a  mixture  of  toxin  and  antitoxin  is  innocuous  to  an  animal,  a 
multiple  of  the  dose,  n  V,  of  the  same  mixture  may  be  toxic  ;  it 
would  also  explain  Buchner's  experiments  alluded  to  above  (p.  161), 
and  Roux's  experiments  in  which  a  toxin-antitoxin  mixture  in- 
nocuous to  normal  guinea-pigs  was  toxic  to  guinea-pigs  whose 
resistance  had  been  reduced  by  injections  of  the  Massowah  vibrio. 

Nernst  has  questioned  from  the  mathematical  standpoint  the 
validity  of  the  views  of  Arrhenius,  and  so  has  Craw  from  much 
experimental  work  on  agglutination  and  on  the  interaction  between 
megateriolysin  and  anti-megateriolysin  ;  Craw  also  considers  that 
there  is  some  doubt  attaching  to  Arrhenius's  calculations.  Accord- 
ing to  Craw,  the  two  substances  most  thoroughly  investigated  by 
Arrhenius  and  Madsen,  diphtheria  toxin  and  tetanolysin,  do  not 
admit  of  sufficiently  exact  determination,  the  former  because  of  the 
uncertainty  attaching  to  animal  experiments,  the  latter  because 
tetanolysin  is  a  most  unstable  body.  Working  with  a  more  stable 
substance,  megateriolysin,  he  holds  that  the  Arrhenius  and  Madsen 
equation  does  not  apply.  Again,  on  the  addition  of  a  small  amount 
of  antitoxin  to  toxin  there  is  no  decrease  in  toxicity  (as  noted  by 
Ehrlich  and  attributed  by  him  to  the  presence  of  toxoid)  as  there 
should  be,  and  Arrhenius  was  thus  forced  to  the  conclusion  that  a 
second  substance,  epitoxonoid,  is  present  with  the  toxin  in  diphtheria 
toxin.  Craw  denies  that  the  toxin-antitoxin  reaction  is  reversible, 
believes  that  antitoxin  must  be  regarded  as  a  colloid  (and  is  not  in 
true  solution),  that  the  mixture  therefore  is  heterogeneous,  not 
homogeneous,  and  that  the  chemical  law  of  mass  action  is  not 
applicable. 

On  the  other  hand,  Craw  maintains  that  the  phenomena  of  the 
toxin-antitoxin  reaction,  including  the  Danysz  effect,  have  their 
counterpart  in  adsorption  phenomena,  such  as  occur  in  the  staining 


ADSORPTION  167 

of  paper,  porcelain,  etc.,  with  anilin  dyes,  in  the  "  adsorption  "  of 
substances  by  colloids,  etc.,1  and  this  view  is  supported  by  Bordet 
and  Gengou.  Thus,  when  solutions  of  arsenious  acid  are  shaken 
up  with  colloidal  ferric  hydroxide,  a  portion  of  the  arsenic  is  taken 
up  by  the  ferric  hydroxide  and  a  portion  remains  in  solution. 
Moreover,  more  arsenious  oxide  is  taken  up  by  the  ferric  hydroxide 
from  dilute  than  from  concentrated  solutions  ;  this  has  its  counter- 
part in  agglutination.  Again,  when  an  antitoxin  is  added  to  a 
toxin  in  just  sufficient  amount  to  produce  a  non- toxic  solution,  the 
amount  of  toxin  which  must  then  be  added  to  constitute  a  fatal 
dose  is  greater  than  the  minimum  lethal  dose  without  antitoxin. 
This  is  also  found  to  be  the  case  with  ferric  hydroxide  and  arsenious 
acid  ;  if  ferric  hydroxide  and  arsenious  acid  are  mixed  so  as  to  form 
just  a  non-toxic  mixture,  the  amount  of  arsenious  acid  which  must 
then  be  added  to  render  the  mixture  toxic  is  greater  than  the  toxic 
dose  of  arsenious  acid.2 

If  pieces  of  filter-paper  be  placed  in  a  dilute  solution  of  stain  at 
sufficiently  long  intervals,  the  pieces  first  immersed  will  become 
coloured  while  those  last  immersed  will  remain  colourless.  On  the 
other  hand,  if  all  the  pieces  be  simultaneously  placed  in  the  solution 
they  all  become  coloured  to  the  same  degree.  This  is  exactly 
comparable  to  the  Danysz  effect.  All  the  phenomena  of  the  toxin- 
antitoxin  reaction  seem  best  explained  on  the  adsorption  hypothesis 
of  Bordet.  Specificity,  it  is  true,  is  not  completely  explained 
thereby,  nor  is  it  explained  by  any  other  hypothesis.3 

The  antitoxic  constituent  of  antitoxin  seems  to  be  a  protein 
body,  probably  allied  to  globulin,  and,  as  already  mentioned,  the 
globulin  content  of  the  blood  of  an  animal  treated  for  antitoxin 
production  increases  in  some  cases.  Tizzoni,  by  precipitating  the 
antitoxic  serum  by  saturation  with  magnesium  sulphate  at  30°  C., 
obtained  the  antitoxin  in  the  precipitate.  By  partial  saturation  of 
antitoxic  serum  with  ammonium  sulphate,  the  antitoxin  is  carried 
down  with  the  second  precipitate,  that  is,  with  the  pseudo-globulin 

1  "  Adsorption  "  is  physical  in  nature  and  mainly  due  to  surface 
condensation. 

2  See  Findlay,  Physical  Chemistry  and  its  Applications  in  Medical 
and  Biological  Science,  1905. 

3  On  the  toxin-antitoxin  reaction  see  Craw,  Proc.  Roy.  Soc.  Lond., 
B.  vol.  Ixxvi,  1905,  p.  179  ;  Journ.  of  Hyg.,  vol.  vii,  1907,  p.  501  ;   and 
ibid.  vol.  ix,  1909,  p.  46 ;    Arrhenius,  Immuno-chemistry,   1907,    and 
Journ.  of  Hyg.,  vol.  viii,  1908,  p.  1  ;   Madsen,  Brit.  Med.  Journ.,  1904, 
vol.  ii,  p.  567  ;  Bordet,  Ann.  de  VInst.  Pasteur,  xvii,  p.  161  ;  McKendrick, 
Proc.  Eoy.  Soc.  Lond.,  B,  vol.  Ixxxiii,  1911,  p.  493  ;   Gengou,  Journ.  of 
State  Med^  xx,  1912,  pp.  65  and  141  (Bibliog.) 


168  A  MANUAL  OF  BACTERIOLOGY 

fraction.  It  is  thus  possible  to  concentrate  antitoxic  serum  and  to 
make  use  of  a  weak  serum,  which  would  otherwise  be  inconvenient 
on  account  of  the  volume  necessary  to  inject  in  order  to  introduce 
the  requisite  amount  of  antitoxin.  For  this  purpose  various  salts 
have  been  employed  for  saturation,  ammonium  sulphate  (Pick  and 
others),  magnesium  sulphate  (Dieudonne),  mixtures  of  sodium  and 
potassium  chlorides  (Atkinson),  etc. 

Dzergowski  and  Predtechensky  l  have  elaborated  a  very  exact 
method  by  which  they  state  that  the  whole  of  the  antitoxin  can  be 
concentrated  and  recovered  from  a  comparatively  weak  serum  by 
means  of  precipitation  with  ammonium  sulphate. 

ANAPHYLAXIS. — An  animal  usually  becomes  more  and 
more  tolerant  to  injections  of  an  antigen,  e.g.  to  diphtheria 
and  tetanus  toxins  in  the  preparation  of  the  corresponding 
antitoxins.  Sometimes,  however,  the  opposite  effect  is 
produced,  viz.  increased  sensitiveness.  This  has  been 
noticed  in  the  preparation  of  tetanus  antitoxin ;  after 
the  animal  has  received  a  few  doses  of  the  toxin  without 
ill-effect,  a  smaller  dose  of  toxin  may  cause  fatal  tetanus. 
The  tuberculin  reaction  is,  probably,  another  example  ; 
tubercle  toxins  circulating  in  the  tuberculous  individual 
render  him  peculiarly  sensitive  to  a  minute  dose  of  tuber- 
culin (i.e.  tubercle  toxin)  which  in  a  normal  person  produces 
no  effect.  Sensitisation  may  be  obtained  with  difficulty 
by  administration  by  the  mouth,  and  this  may  be  the 
explanation  of  the  urticaria,  etc.,  produced  in  some  indi- 
viduals by  certain  foods,  e.g.  shell-fish.  This  condition  of 
hypersensitiveness  is  known  as  "  anaphylaxis  "  (i.e.  the 
opposite  of  "  prophylaxis  ").  Probably  any  antigen  under 
particular  conditions  may  induce  anaphylaxis,  but  the 
phenomenon  has  been  especially  studied  in  connexion  with 
serum  injections,  though  any  protein,  e.g.  egg-white  or 
bacterial  cells,  similarly  causes  it.  The  injection  of  an 
anti- serum  usually  produces  no  ill- effect  other  than  the 
rashes,  joint  pains,  and  pyrexia  already  mentioned,  even 

1  .See  Hewlett's  Serum  Therapy,  1910,  p.  68. 


ANAPHYLACTTC  SHOCK  169 

if  large  amounts  of  the  serum  be  given  extending  over  days 
or  even  weeks,  but  a  second  injection  of  serum  given  after 
a  first  injection  with  an  interval  of  twelve  days  or  more 
between  the  two  injections  is  liable  to  be  followed  by 
effects  which  may  be  more  or  less  serious,  constituting 
the  so-called  "  anaphy lactic  shock  "  or  "  serum  disease," 
or  immediate  or  accelerated  reactions,  "  supersensitisation," 
may  ensue  (see  p.  161). 

In  anaphy  lactic  shock,  plain  muscle  contracts  and  Dale  1 
has  used  the  excised  uterus  of  sensitised  guinea-pigs  to 
give  a  graphic  record  of  the  action  of  the  reacting  dose. 
Specificity  is  shown  by  the  fact  that  the  uterus  of  a  guinea- 
pig  sensitised  with  sheep-serum  contracts  only  when  flooded 
with  a  reacting  dose  of  sheep-serum  and  not  with  any 
other  serum.  The  animal  may  be  sensitised  with  two  or 
three  different  proteins  and  then  the  uterus  contracts  in 
turn  to  each  reacting  dose  of  the  different  proteins.  Once 
the  reacting  dose  has  been  given  and  the  uterus  has  con- 
tracted, the  muscle  is  no  longer  sensitive  to  the  protein. 

The  symptoms  of  anaphylactic  shock  are  nausea  and 
vomiting,  small  and  rapid  pulse,  faintness  or  more  serious 
heart  failure,  dyspnoea  with  rapid  and  shallow  respiration 
and  feeling  of  suffocation,  collapse,  rigors,  convulsions, 
and  even  coma.  The  severity  of  the  symptoms  varies  in 
different  cases,  and  the  symptoms  usually  pass  off  in  the 
course  of  an  hour  or  two  ;  but  a  few  fatal  cases  have  been 
recorded.  Death  is  easily  produced  experimentally,  and, 
post-mortem,  scattered  ecchymoses  are  found  and  a  dis- 
tended condition  of  the  lungs  due  to  spasm  and  contraction 
of  the  bronchioles,  to  which  the  fatal  event  is  due. 

In  the  immediate  reaction,  rash,  pyrexia,  joint  pains, 
vomiting,  rigors,  and  occasionally  convulsions  and  collapse 
occur,  generally  within  six  hours  after  the  second  injection 
of  serum.  In  the  accelerated  reaction,  these  phenomena 

1  Jmirn.  Pharmacd.  and  Exp*r.  Therapeutics,  IV,  1913-14,  p.  167. 


170  A  MANUAL  OF  BACTERIOLOGY 

appear  between  the  eighteenth  hour  and  the  fifth  day  after 
the  second  injection  of  serum. 

The  immediate  and  accelerated  reactions  may  occur  a 
long  time  after  the  first  course  of  serum  treatment  if  more 
serum  be  given.  Goodall  records  one  case  in  which  over 
four  years  elapsed  between  serum  treatments  for  first 
and  second  attacks  of  diphtheria,  an  accelerated  reaction 
occurring  after  the  reinoculation  for  the  second  attack. 

The  amount  of  serum  given  does  not  definitely  influence 
the  result.  The  remarkable  features  of  the  phenomenon 
are — (1)  they  do  not  occur  unless  an  interval  of  about 
twelve  days  or  more  elapses  between  the  two  injections 
of  serum  ;  (2)  the  long  period  which  may  intervene  between 
the  two  injections  of  serum  and  still  be  accompanied  by 
symptoms  ;  (3)  the  serious  nature  of  the  condition  in 
some  instances. 

The  explanation  of  the  phenomenon  is  difficult.  Un- 
doubtedly the  symptoms  are  due  to  some  substance  in 
the  serum  which  has  a  toxic  action,  and  have  nothing 
to  do  with  the  antitoxic  constituent,  for  normal  serum 
produces  the  same  effects. 

In  experimental  anaphylaxis  produced  in  animals  by 
the  injection  of  normal  serum,  it  is  found  that  the  con- 
dition only  occurs  if  the  two  doses  of  serum  are  separated 
by  an  interval  of  about  twelve  days  or  more ;  the  first  is 
termed  the  sensitising,  the  second  the  reacting,  dose.  The 
larger  the  sensitising  dose,  the  longer  must  the  interval 
be  for  the  reacting  dose  to  produce  a  maximum  effect. 
Moreover,  the  two  injections  must  be  of  the  same  serum 
or  other  protein ;  thus  a  first  injection  of  horse  serum 
followed  by  a  second  injection  of  rabbit  serum  would 
not  produce  it.  Extremely  small  doses  of  serum  will 
also  bring  it  about ;  and  lastly,  ansesthetisation,  when 
the  second  dose  of  serum  is  given,  prevents  the  develop- 
ment of  the  symptoms — a  very  extraordinary  result. 


ANAPHYLAXIS  171 

The  Arthus  phenomenon  occurs  when  a  guinea-pig 
receives  several  doses  of  normal  horse  serum  at  intervals 
of  some  days.  Another  injection  of  horse  serum  then 
causes  an  cedematous  mass,  an  aseptic  abscess,  or  an  area 
of  necrosis  at  the  site  of  the  new  inoculation,  which  may 
be  far  removed  from  the  region  of  the  previous  inoculations, 
and  the  animal  becomes  cachectic  and  dies. 

The  Theobald  Smith  phenomenon  occurs  when  a  guinea- 
pig  has  been  sensitised  by  a  very  small  single  dose  of  normal 
horse  serum,  0-01  c.c.,  0-001  c.c.,  or  even  0-000001  c.c.  ; 
if,  then,  after  an  interval  of  twelve  to  fourteen  days  a 
somewhat  larger  dose  of  serum,  0-1  c.c.,  be  given,  the  serious 
symptoms  of  hypersensitiveness  develop  within  a  few 
minutes,  viz.  respiratory  failure,  paralysis,  clonic  spasms, 
and  frequently  death.  At  one  time  it  was  believed  that 
a  small  sensitising  dose  is  more  effective  than  a  large  one 
in  producing  anaphylactic  shock,  but  it  has  been  shown 
that  this  is  not  the  case,  a  large  dose  merely  lengthens  the 
incubation  period  (up  to,  it  may  be,  forty  days).  The 
reason  for  this  may  be  that  the  toxic  substance  slowly 
formed  by  the  sensitising  dose  combines  as  it  is  produced 
with  a  part  of  the  antigen  injected,  so  that  the  ultimate 
result  is  as  though  a  small  sensitising  dose  had  been  injected. 

Various  hypotheses  have  been  advanced  to  account  for 
anaphylaxis.  The  fact  that  an  interval  or  incubation 
period  is  necessary  for  the  development  of  the  condition 
clearly  points  to  the  formation  of  anti- bodies  as  a  necessary 
part  of  the  phenomenon.  Moreover,  a  "  passive  "  anaphy- 
lactic condition  may  be  induced  in  an  animal  by  injecting 
it  with  the  serum  of  a  sensitised  animal :  this  treated 
animal  suffers  from  anaphylactic  shock  on  being  injected 
with  the  antigen.  The  substance  which  gives  rise  to  the 
anaphylactic  shock  is  termed  "  anaphylatoxin "  by 
Friedberger  and  "  apotoxin  "  by  Richet. 

Besredka  believes  that  anaphylaxis  is  caused  by  the 


172  A  MANUAL  OF  BACTERIOLOGY 

presence  of  two  substances  in  the  serum,  one  thermostable 
and  having  the  properties  of  an  antigen  (see  p.  150),  which 
he  terms  "  sensibilisogen/'  and  which  on  injection  produces 
its  anti-body,  "  sensibilisin."  The  other  substance  is 
thermolabile,  and  is  termed  "  anti-sensibilisin,"  and 
combines  with  sensibilisin  whenever  it  meets  with  the 
latter.  Sensibilisin  is  particularly  fixed  by  the  cells  of  the 
nervous  system,  and,  according  to  Besredka,  it  is  the 
violent  reaction  between  anti-sensibilisin  and  sensibilisin 
in  the  nerve  tissues  which  causes  the  serious  disturbance 
characteristic  of  anaphylaxis.  When,  therefore,  a  small 
dose  of  serum  (po,,-.-,1,,  c.c.)  is  administered,  the  sensibili- 
sogen  slowly  forms  sensibilisin.  If  a  second  dose  of  serum 
is  given  twelve  days  or  more  after  the  first  injection,  the 
anti-sensibilisin  in  it  combines  with  the  sensibilisin  formed 
by  the  first  injection,  and  disturbance  results. 

The  reason  why  ana3sthetisation  with  ether  when  the 
second  injection  is  given  prevents  the  symptoms  of  ana- 
phylaxis developing  is  that  the  anaesthetic  renders  the 
nerve  cells  insensitive  to  the  reaction  between  the 
sensibilisin  and  antisensibilisin. 

According  to  Richet,  a  "  toxigen  "  is  formed  in  the  blood 
or  cells  at  the  end  of  the  incubation  period  and  persists 
for  a  long  period.  A  toxic  apotoxin  or  precipitin  is  formed 
as  a  result  of  the  interaction  of  toxigen  with  antigen,  the 
toxicity  of  which  is  further  increased  by  combination  with 
the  alexin  of  the  blood. 

Bordet  suggests  that  the  union  of  anti-body  and  antigen 
creates  a  complex  which  by  adsorption  monopolises  certain 
principles  in  the  blood  plasma  which  then  becomes  toxic. 
Thus  Wassermann  and  Reysser  found  that  if  guinea-pig 
serum  and  kaolin,  *an  inert  powder,  be  mixed  and  then 
centrifuged,  the  intravenous  injection  of  the  fluid  is 
followed  by  symptoms  closely  resembling  those  of  anaphy- 
laxis. A  weak  agar  jelly  (0-05  per  cent.)  acts  similarly. 


ANTI-MICROBIC  SERA  173 

The  serum  must  be  fresh  and  active  ;    serum  heated  to 
56°  C.  is  inert. 

Anaphylaxis,  supersensitisation,  or  hypersensitisation 
may  be  of  considerable  importance  in  serum  treatment. 

On  the  serum  disease,  supersensitisation,  and  anaphylaxis,  see 
Hewlett,  Serum  Therapy,  ed.  2,  1910  ;  Rosenau  and  Anderson, 
Journ.  Amer.  Med.  Assoc.,  1906,  p.  1007  ;  Von  Pirquet  and  Schick, 
Die  Serum-Krankheit,  1905  ;  Richet,  Ann.  de  Vlnst.  Pasteur,  xxi, 
p.  497,  and  Anaphylaxis  (Constable  and  Co.,  1913.  Bibliog.)  ; 
Besredka,  Ann.  de  Vlnst.  Pasteur,  xxi,  p.  950,  and  Bull,  de  Vlnst. 
Pasteur,  vii,  1909,  p.  721  ;  Currie,  Journ.  of  Hygiene,  vol.  vii,  1907, 
pp.  35,  61,  and  vol.  viii,  1908,  p.  457  ;  Grunbaum,  ibid.  vol.  viii, 
1908,  p.  9  ;  Goodall,  ibid.  vol.  vii,  1907,  p.  607  ;  Bordet,  Journ. 
State  Med.,  1913,  p.  449.1 

ANTI-MICROBIC  SERA. — If  an  animal  be  injected  with 
increasing  doses  of  bacteria,  care  being  taken  to  keep 
below  a  lethal  one,  the  animal  gradually  becomes  accus- 
tomed to  the  microbe,  and  ultimately  acquires  a  high 
degree  of  immunity,  so  that  it  is  unaffected  by  amounts 
which  would  infallibly  kill  an  untreated  animal.  More- 
over, the  blood-serum  of  such  a  treated  animal,  if  injected 
into  a  second  animal,  will  protect  the  latter  against  a  few 
lethal  doses  of  the  microbe,  but  not  against  a  large  amount. 
Nor  is  the  protection  afforded  proportional  to  the  amount 
of  serum  injected  ;  for  example,  if  0-005  c.c.  of  anti-cholera 
serum  will  protect  against  5  mgrm.  of  living  cholera  culture, 
three  times  as  much,  or  0-015  c.c.  of  the  serum,  will  not 
protect  against  15  mgrm.  of  cholera  culture,  and  when  a 
certain  dose  of  the  culture  is  reached  no  amount  of  serum 
will  save  the  animal.  The  mode  in  which  the  serum  acts 
may  be  studied  microscopically.  If  cholera  anti-serum 
and  cholera  culture  be  injected  into  the  peritoneal  cavity 
of  a  guinea-pig,  and  the  peritoneal  contents  be  examined 
at  short  intervals  afterwards,  it  will  be  found  that  the 

1  Trans.  XVIIthlntcrnat.  Cong,  of  Medicine,  1913,  Sect.  IV,  Pt.  I,  pp.  1 
(Bobi-cdka)  and  13  (Richet),  and  ibid.  Pt.  II. 


174  A  MANUAL  OF  BACTERIOLOGY 

vibrios  lose  their  motility,  become  distorted  and  globular, 
undergo  solution,  and  finally  disappear.  The  protection 
afforded  by  the  anti-serum  is  therefore  due  to  the 
destruction  of  the  microbes  by  solution,  the  process 
being  known  as  bacteriolysis,1  and  the  bodies  which  bring 
it  about  being  termed  "  bacteriolysins."  The  reaction  is 
known  as  "  Pfeiffer's  phenomenon  "  or  reaction,  from  its 
discoverer.  If  the  serum  and  the  microbes  be  mixed  in 
vitro  the  latter  are  unaffected ;  apparently,  therefore, 
some  constituent  of  the  living  body  in  addition  to  the 
anti-serum  is  necessary  for  the  solution  of  the  microbes. 
But  in  1895  Metchnikoff  showed  that  the  reaction  will 
take  place  in  vitro  provided  that  some  of  the  fresh  peri- 
toneal exudate  of  a  normal  guinea-pig  be  added  to  the 
mixture  of  anti-serum  and  microbes.  The  same  year 
Bordet  found  that  the  addition  of  the  peritoneal  exudate 
is  unnecessary  provided  the  anti- serum  be  perfectly  fresh. 
These  experiments  prove  that  the  solution  of  the  microbes 
is  brought  about  by  the  interaction  of  at  least  two  sub- 
stances, one  of  which  is  present  in  all  fresh  serum  and  in 
the  living  body,  but  is  unstable,  disappearing  on  keeping 
or  heating  the  serum,  the  other  is  a  relatively  stable  body 
produced  during  the  process  of  inoculation.  The  former, 
the  unstable  normal  body  present  in  all  animals,  is  usually 
termed  "  complement "  (Ehrlich  and  Morgenroth),  "  alexin  " 
(Buchner  and  Bordet),  or  "  addiment  "  ;  while  the  stable 
constituent  produced  by  immunisation  is  known  as  the 
"  amboceptor  "  (Ehrlich),  "  immune  body,"  "  interme- 
diary," "preparer"  (Gruber),  "  fixateur  "  (Metchnikoff), 
or  "  substance  sensibilisatrice  "  (Bordet). 

These  considerations  suggest  an  explanation  why  anti-microbic 
serum  neutralises  only  a  limited  amount  of  living  culture,  viz.  the 
amount  of  complement  present  in  the  body  at  one  time  is  limited, 
and  when  this  has  been  used  up  bacteriolysis  ceases.  Anti -micro bic 
sera  are  relatively  inefficient  in  practice,  insufficiency  of  complement 

1  See  Gruber,  "  Harbcu  Lectures,"  Journ.  State  Med.,  1902. 


AMBOCEPTOR  AND  COMPLEMENT          175 

being  suggested  as  the  reason.  Attempts  have  been  made  to  supple- 
ment the  complement  present  by  injecting  fresh  normal  serum  with 
the  anti-serum,  but  without  success,  and  some  anti-micro bic  sera, 
e.g.  anthrax  serum,  are  not  bacteriolytic  ;  this  explanation  is,  there- 
fore, unsatisfactory.  Deflection  of  complement  (p.  178)  may  occur 
in  some  instances,  or  the  complement  may  not  be  of  the  right  kind. 
In  other  cases,  the  organism  in  certain  situations  may  be  inaccessible 
to  the  blood-stream  and  to  the  anti -serum, 
e.g.  the  vibrios,  in  the  bowel  in  cholera. 

Another  reason  advanced  is  the  extreme 
specificity  of  anti-serum  and  the  variability 
of  bacteria  so  that  many  races  or  strains 
of  an  organism  may  exist,  e.g.  of  B.  coli, 
streptococci,  pneumococci,  etc.  Hence  the 
anti-serum  prepared  with  one  race  may  not 
neutralise  another  race.  Attempts  have 
been  made  to  overcome  this  factor  by  pre- 
paring the  anti-serum  by  the  injection  of 
many  races  and  so  obtaining  a  "  polyvalent 
serum." 

FIG.    33.— Diagram   to 

The  amboceptor  or  immune  body  show  the  union  be- 
seems to  link  the  complement  to  the  *™ee"  complement 

(black)     and     proto- 

bactermm  (Fig.  33) ;   complement  re-  piasm    of    cell    by 

mains  free  if  the  appropriate  ambo-  means  of  the  ambo- 

u  j     •  ceptor  (white).  (After 

ceptor  or  immune  body  is  not  present,  Ehrlich ) 

and  bacteriolysis  does  not  ensue  (see 
also  p.  174).     Complement  is  thermolabik,  i.e.  it  is  destroyed 
by  heating  to  56°  C.  for  thirty  minutes ;    while  the  ambo- 
ceptor  is    thermostable,   i.e.  it  is  not  destroyed  by  this 
treatment. 

According  to  Ehrlich,  fresh  serum  contains  numerous 
complements  which  are  more  or  less  specific  for  different 
amboceptors  (see  also  note,  p.  182).  When  the  comple- 
ment is  destroyed  by  heating  it  is  converted  into  "  comple- 
mentoid  "  (analogous  to  toxoid).  Both  complement  and 
complementoid  on  injection  give  rise  to  anti- complement. 
The  amount  of  complement  in  different  sera  varies  con- 
siderably ;  horse  serum  contains  very  little,  guinea-pig 
seruni  much.  Complement  itself  probably  consists  of  two 


176  A  MANUAL  OF  BACTERIOLOGY 

portions,  as  it  is  generally  accepted  that  it  can  be  split 
into  a  "  mid-piece  "  and  an  "  end-piece  "  by  the  action  of 
dilute  hydrochloric  acid,  carbon  dioxide,  and  dialysis. 
The  mid-piece  is  thought  to  be  in  the  globulin  fraction, 
the  end-piece  in  the  albumin  fraction.  Noguchi,  however, 
considers  that  the  whole  complement  is  present  in  the 
albumin  fraction  and  that  inactivation  of  the  complement 
by  acid,  etc.,  is  due  not  to  splitting  into  two  fractions,  but 
to  inactivation  of  the  whole  complement. 

Pfeiffer's  reaction  is  of  considerable  value  in  practical 
bacteriology  for  the  exact  recognition  of  bacterial  species. 
A  mixture  of  a  suspension  of  the  organism  to  be  tested  with 
a  small  quantity  of  serum  from  a  highly  immunised  animal 
is  injected  into  the  peritoneal  cavity  of  a  normal  guinea- 
pig.  The  fluid  in  the  peritoneal  cavity  is  then  examined 
microscopically  half  to  one  hour  after  the  injection,  and 
if  the  reaction  be  positive  the  organisms  will  be  found  in 
all  stages  of  degeneration,  being  mostly  converted  into 
spherules.  In  this  case,  according  to  Pfeiffer,  the  organism 
is  to  be  regarded  as  belonging  to  the  same  species  as  that 
by  means  of  which  the  immunisation  of  the  animal,  from 
which  the  blood-serum  was  obtained,  was  carried  out.  If, 
on  the  other  hand,  the  reaction  be  negative,  the  organisms 
are  unaffected  after  being  in  the  peritoneal  cavity  for  an 
hour  or  so,  and  the  organism  is  then  considered  to  be  a 
species  different  from  that  used  for  the  immunisation. 
Thus,  Pfeiffer's  reaction  may  be  made  use  of  to  differentiate 
the  cholera-like  vibrios  from  true  cholera  vibrios  and  the 
members  of  the  typhoid- colon  group  from  one  another. 

The  destruction  of  the  bacteria  by  bacteriolysis  is 
regarded  by  some  as  being  brought  about  by  osmotic 
changes,  by  others  by  processes  analogous  to  digestion. 
During  bacteriolysis  the  specific  immunising  substances 
and  anti-bodies  are  used  up,  and  for  the  lysis  of  a  given 
quantity  of  bacteria  a  certain  amount  of  immune  serum 


PFEIFFER'S  REACTION  177 

is  necessary,  while  after  lysis  has  taken  place  the  latter 
loses  the  power  of  dissolving  bacteria.  The  same  holds 
good  for  haemolysis,  and  the  facts  relating  to  bacteriolysis 
and  haemolysis  are  almost  interchangeable. 

Anti-endotoxic  sera. — The  comparative  inefficiency  of  anti- 
microbic  sera,  particularly  typhoid,  led  Macfadyen  to  attempt  to 
prepare  sera  with  microbial  endotoxins,  and  the  work  has  been 
continued  by  Siidmersen  and  the  writer.  The  method  was  to 
immunise  horses  with  the  endotoxin  obtained  by  the  method 
described  on  p.  40.  With  a  typhoid  serum  so  prepared  Goodall 
and  the  writer  obtained  promising  results.1 

Method  of  applying  Pfeiffer's  reaction. — For  Pfeiffer's  test,  the 
organism  must  be  virulent,  and  a  high-grade  immune  serum  is 
necessary.  If  the  organism  is  not  virulent,  it  is  spontaneously 
destroyed  in  the  peritoneal  cavity  without  the  addition  of  immune 
serum.  The  method  may  be  best  explained  in  the  case  of  a  vibrio 
supposed  to  be  the  cholera  vibrio.  The  cholera-immune  serum 
(obtained  from  a  horse  repeatedly  injected  with  cholera  culture) 
should  possess  a  titre  of  not  less  than  0-0002  c.c.,  i.e.  this  amount 
of  serum  mixed  with  one  loop  (2  mgrm.)  of  an  eighteen-hour  agar 
cholera  culture  (virulent),  suspended  in  1  c.c  of  broth,  and  injected 
into  the  peritoneal  cavity  of  a  small  guinea-pig  should  cause  granular 
degeneration  and  bacteriolysis  of  the  vibrios  within  one  hour. 

Four  mixtures  are  made — (a)  one  loop  of  an  eighteen-hour  agar 
culture  of  the  vibrio  to  be  tested,  0-001  c.c.  cholera-immune  serum, 
suspended  in  1  c.c.  of  broth  ;  (&)  the  same  as  (a),  but  0-002  c.c. 
cholera  serum  ;  (c)  the  same  as  (a),  but  0-001  normal  serum  of  an 
animal  of  the  same  species  as  that  furnishing  the  cholera  serum  ; 
(d)  one  quarter  loop  of  the  vibrio  in  1  c.c.  of  broth,  as  a  control  of 
the  virulence  of  the  culture.  These  mixtures  are  then  injected  into 
the  peritoneal  cavities  of  four  guinea-pigs  each  of  about  250  grin, 
weight.  At  intervals  of  thirty  and  sixty  minutes  hanging-drop 
preparations  are  made  of  the  peritoneal  fluid  of  each  animal,  the 
fluid  being  obtained  by  inserting  a  capillary  pipette  through  a 
minute  incision  in  the  skin.  In  the  guinea-pigs  injected  with  (a) 
and  (6),  if  the  organism  be  cholera,  the  vibrios  should  show  marked 
degenerative  changes  within  sixty  minutes,  while  (c)  and  (d)  will 
show  plenty  of  active  vibrios.  If  the  organism  be  non-virulent, 
two  methods  may  be  adopted  for  applying  the  Pfeiffer  reaction. 
The  first,  a  microscopical  or  direct  method,  is  carried  out  by  micro- 

1  Proc.  Roy.  Soc.  Med.,  vol.  ii,  1907-8,  Med,  Sect.,  p.  245  et  seq. 

12 


178 


A  MANUAL  OF  BACTERIOLOGY 


scopical  examination  of  hanging-drop  specimens  of  the  organism 
suspended  in  a  drop  of  the  immune  serum  to  which  a  trace  of  fresh 
peritoneal  fluid  (complement)  is  added.  If  the  organism  is  homolo- 
gous with  the  immune  serum,  the  bacteria  are  soon  transformed 
into  granules.  Controls  are  put  up  at  the  same  time  with  a  known 
strain  of  the  organism  with  (1 )  its  homologous  immune  serum  -f-  com- 
plement ;  (2)  non-immune  serum 
of  the  same  animal  -f-  comple- 
ment ;  also  of  the  organism  being 
tested  with  non-immune  serum  of 
the  same  animal  -j-  complement. 
The  peritoneal  fluid  may  be  ob- 
tained by  injecting  3-4  c.c.  of 
broth  into  the  peritoneal  fluid 
of  a  guinea-pig  and  four  hours 
later  withdrawing  the  fluid  (now 
turbid  with  leucocytes)  and  oen- 
trifuging,  or  allowing  it  to 
stand  on  ice  for  twenty-four 
hours. 

In  the  second,  or  indirect, 
method,  the  organism  is  used  to 
prepare  an  immune  serum  by 
injecting  an  animal  (e.g.  a  rabbit) 
with  it,  and  the  immune  serum 
so  prepared  is  tested  on  a  known 
virulent  stain  in  the  peritoneal 

cavity  of  guinea-pigs  in  order  to  ascertain  whether  or  no  it  brings 
about  bacteriolysis,  i.e.  the  Pfeiffer  phenomenon. 

Deflection,  deviation,1  diversion  or  blocking  of  complement. — 
Pfeiffer  in  1895  observed  that  a  large  amount  of  immune  serum 
might  not  protect  an  animal  from  the  cholera  vibrio,  while  a  smaller 
amount  with  the  same  dose  of  vibrio  did  so.  In  1901  Neisser  and 
Wechsberg  demonstrated  an  analogous  reaction  in  vitro.  They 
studied  the  effect  of  a  bacteriolytic  immune  serum  when  varying 
amounts  of  the  inactivated  serum  were  employed.  The  quantity 
ranged  from  0-0005  c.c.  to  1  c.c.  To  each  of  these  amounts  constant 
volumes  of  normal  serum  and  bacterial  suspension  were  added.  No 
bacteriolysis  occurred  when  large  and  small  amounts  of  immune 
serum  were  used,  but  with  medium  amounts  bacteriolysis  was 
complete.  Theyj^explained  this  anomalous  reaction,  the  absence 

1  "  Fixation  of  complement "  (p.  183)  is  frequently  erroneously 
termed  "  deviation  of  complement." 


FIG.  34. — Diagram  to  represent 
the  condition  of  the  blood  in 
which  there  is  an  excess  of 
amboceptors.  The  ambocep- 
tors  (white)  unite  with  both 
complement  (black)  and  re- 
ceptors (dotted),  so  that  the 
receptors  cannot  combine  with 
the  amboceptor-complement 
groups. 


AGGRESSItfS  179 

of  bacteriolysis  with  large  amounts  of  immune  serum,  as  follows  : 
When  the  amboceptors  are  in  large  excess,  a  portion  combines 
with  the  complement,  leaving  some  amboceptors  free,  and  these 
free  amboceptors  then  unite  with  the  receptors  before  the  activated 
amboceptors  (amboceptors  +  complement)  do,  and  thus  the  comple- 
ment-amboceptor  groups  are  rendered  inert.  The  reaction  is 
represented  diagrammatically  in  Fig.  34.  Arrhenius,  however,  does 
not  accept  this  explanation.  He  says  :  "If  we  have  the  compounds 
ea  and  ab  which  may  combine  to  form  the  compound  eab,  the 
formation  of  the  latter  depends  wholly  upon  whether  e  has  a  greater 
affinity  for  ab  than  for  a.  If  not,  then  eab  is  not  formed,  even  if  a 
is  not  present  in  excess."  (a  =  amboceptor,  e  =  microbe,  b  =  com- 
plement.) The  phenomenon  may  be  quite  analogous  with  the 
inhibition  met  with  in  agglutination  (p.  188). 


Aggressins 

Bail  has  discussed  the  question  of  the  relationship  between 
bacteriolysis  and  immunity.  He  argues  that  there  is  apparently 
little  relationship  between  the  bactericidal  properties  of  the  body 
fluids  and  the  immunity  of  an  animal  to  infection  through  bacterio- 
lytic  processes  ;  and  points  out  that  in  rabbits  immunised  against 
anthrax  there  is  no  bacteriolytic  power,  the  bacteria  disappearing 
gradually  as  the  result  of  phagocytic  action  of  cells,  chiefly  marrow- 
cells  ;  that  a  comparison  of  the  sera  of  sheep,  rabbits,  and  cattle 
shows  great  variation  in  their  content  of  immune  body,  though  the 
animals  are  almost  equally  susceptible  to  anthrax  ;  and  that  in 
test-tube  experiments  a  bacteriolytic  serum  is  blocked  when  the 
conditions  are  approximated  to  those  in  the  body  by  the  addition 
of  body  cells  to  the  mixture  ;  the  bactericidal  properties  of  the 
serum  disappear  or  are  greatly  inhibited.  Kruse  suggested  that  for 
infection  to  take  place  the  invading  bacteria  must  elaborate  chemical 
substances  which  so  act  on  the  cells  and  fluids  of  the  invaded  animal 
that  they  overcome  its  natural  resistance  against  infection.  These 
substances  are  considered  by  him  and  Bail  to  be  distinct  from  the 
toxins,  and  are  termed  by  these  writers  "  aggressins. " x  The 
aggressins  are  supposed  to  be  secreted  by  the  living  uninjured 
bacteria  and  not  to  be  extracts,  nor  derived  by  solution,  of  the 
bacteria  ;  they  occur  particularly  in  the  fluids  of  pathological 

1  See  Cenlr.  f.  Bakt.,  Orig.,  xlii,  1906,  pp.  51,  139,  241,  335,  437,  and 
546.  Also  an  excellent  summary  by  Marshall,  Philip'pine  Journ, 
of  Science,  vol.  ii,  1907,  p.  352> 


180  A  MANUAL  OF  BACTERIOLOGY 

oedemas  and  cxudates,  and  may  be  obtained  from  these  by  centri- 
fugation  and  sterilisation  at  low  temperatures.  Bail  believes 
that  the  aggressins  cannot  be  anti-complements,  anti-immune 
bodies,  etc.,  but  are  substances  heretofore  unrecognised  and  the 
active  substances  of  the  infection,  and  he  considers  that  in  order  to 
produce  true  immunity  in  disease  anti-aggressin  sera  must  be 
prepared.  The  following  are  some  of  the  properties  of  these  sup- 
posed aggressins  :  (1)  Sterilised  aggressin  with  a  non-lethal  dose  of 
the  corresponding  organism  renders  the  latter  fatal  ;  (2)  aggressin 
alone  is  only  slowly  toxic,  producing  a  prolonged  illness  with 
emaciation  preceding  death  ;  (3)  inoculation  of  aggressin  with 
bacteriolytic  serum  into  the  peritoneal  cavity  suspends  the  action 
of  the  latter  ;  (4)  aggressin  with  bacteria  blocks  phagocytosis. 
Bail  believes  that  the  aggressins  promote  infection  by  interfering 
with  the  protective  mechanism  of  the  infected  animal,  particularly, 
if  not  solely,  by  inhibiting  phagocytosis.  Upon  the  power  to  pro- 
duce aggressin  Bail  has  classified  bacteria  into  (1)  true  parasites 
which  always  produce  aggressin,  e.g.  anthrax  and  chicken  cholera  ; 
(2)  half -parasites,  the  aggressin-producing  power  of  which  is  variable, 
e.g.  typhoid,  cholera,  dysentery,  and  plague  ;  (3)  saprophytes.  The 
virulence  of  an  organism  does  not  coincide  with  aggressivity,  and 
extremely  virulent  bacteria  may  be  half -parasites. 

Bail's  hypotheses  have  been  much  criticised,  and  Wassermann 
and  Citron  believe  that  the  supposed  aggressins  are  derivatives  of 
the  bacterial  protoplasm  which  have  the  power  of  combining  with 
the  specific  protective  substances  of  the  animal  and  so  inhibit 
the  action  of  the  latter  ;  they  are,  in  fact,  endotoxins  of  feeble 
toxicity. 

HAEMOLYSIS.1 — Some  blood  sera  possess  marked  powers 
of  dissolving  the  red  blood- corpuscles  of  another  species, 
and  of  setting  free  their  contained  hemoglobin  (e.g.  goat 
serum  dissolves  rabbits'  and  guinea-pigs'  corpuscles,  and 
ox  and  human  sera  usually  dissolve  sheep's  corpuscles), 
and  if  an  animal  be  injected  with  the  blood-corpuscles  of 
another  species  its  blood-serum  generally  acquires  the 
property  of  dissolving  the  blood- corpuscles  with  which 

1  See  Bulloch,  Practitioner,  December  1900,  p.  672,  and  Trans.  Path. 
Soc.  Lond.,  vol.  Hi,  Part  3,  1901,  p.  208  ;  Gruber,  "  Harben  Lectures," 
Journ.  State  Med.,  1902,  February,  March,  and  April ;  Ehrlich,  Collected 
Studies  on  Immunity  ;  Muir,  Studies  on  Immunity. 


HAEMOLYSIS  181 

it  has  been  injected.  For  example,  the  serum  of  a  normal 
rabbit  has  no  haemolytic  action  upon  the  red  corpuscles 
of  the  sheep  ;  but  if  a  rabbit  receive  a  few  injections  of 
defibrinated  sheep's  blood,  its  blood-serum  acquires 
haemolytic  properties  and  dissolves  the  red  corpuscles  of 
the  sheep.  This  solution  of  the  blood- corpuscles  is  termed 
"  haemolysis,"  and  the  substances  which  produce  haemo- 
lysis are  "  haemolysins."  If  the  active  serum  be  heated  to 
56°  C.  it  is  "  inactivated  "  and  loses  its  haemolysing 
power,  but  can  again  be  rendered  haemolytic  or  "  acti- 
vated "  by  the  addition  of  fresh  normal  serum ;  normal 
serum,  however,  rapidly  loses  its  activating  properties 
on  keeping.  It  will  thus  be  seen  that  there  is  an  almost 
complete  analogy  between  bacteriolysis  and  haemolysis, 
the  latter  being  brought  about  by  the  interaction  of  two 
substances,  one  specific  and  stable  produced  by  the  injec- 
tions, the  haemolytic  "  amboceptor  "  or  "  immune  body," 
and  the  other  an  unstable  body  present  in  fresh  normal 
serum,  the  "  complement  "  or  "  alexin." 

Haemolysin  formed  by  the  injection  of  corpuscles  of 
another  species  is  termed  "  heterolysin."  If  corpuscles 
of  the  same  species  be  injected,  haemolysin  is  formed 
("  isolysin  "),  but  the  injection  of  the  animal's  own  cor- 
puscles does  not  give  rise  to  haemolysin,  i.e.  "  autolysin  " 
is  not  formed. 

Blood- corpuscles  are  more  tangible  entities  than  bac- 
teria, and  are  far  easier  to  work  with  than  the  latter,  and 
haemolysis  has  been  the  subject  of  a  large  amount  of 
experimental  work  by  Bordet  and  Gengou,  Ehrlich,  Mor- 
genroth,  Gruber,  Bulloch,  Muir,  and  others,  and  the  results 
obtained  have  shed  considerable  light  upon  the  complex 
phenomena  of  immunity  and  of  the  actions  of  anti- bodies 
in  general.  Moreover,  the  globulicidal  material  in  haemo- 
lysis seems  to  be  identical  with  the  bactericidal  one  in 
bacteriolysis — that  is  to  say,  it  is  the  complement  or 


182  A  MANUAL  OF  BACTERIOLOGY 

alexin. x  According  to  Ehrlich's  view,  whether  it  be  normal 
or  "  immune  "  serum  (i.e.  serum  of  a  treated  animal), 
bacteriolysis  or  haemolysis  takes  place  only  when  the 
complement  and  amboceptor  unite  (Fig.  33,  p.  175), 
complement  by  itself  having  little  affinity  for  the  bacterium 
or  erythrocyte,  the  combination  forming  the  "  lysin," 
which  then  acts.  According  to  Gruber,  however,  neither 
bacteriolysin  nor  hsemolysin  exist  as  a  chemical  entity, 
the  specific  bacteriolytic  or  hsemolytic  action  being  due 
to  the  fact  that  the  cells  first  absorb  the  amboceptor  and 
so  become  accessible  to  the  complement,  for  the  two 
substances  do  not  combine  in  definite  proportions — the 
more  the  blood- corpuscles  are  laden  with  the  amboceptor 
the  smaller  the  quantity  of  complement  required  to  bring 
about  their  solution. 

Many  bacteria — e.g.  B.  pyocyaneus,  B.  typliosus,  staphy- 
lococci  and  streptococci — produce  hsemolysins,  and  the 
haemoglobin  staining  occurring  in  septic  diseases,  etc.,  is 
probably  partly  due  to  the  action  of  bodies  of  this  nature 
elaborated  by  the  infecting  organisms. 


Practical  Uses  of  Haemolysis,  etc. 

1.  Haemolysis  test. — Some  micro-organisms  produce  non-specific 
hsemolysins,  others  do  not ;  this  may  constitute  a  difference  between 
allied  organisms.  For  instance,  as  a  rule  true  cholera  vibrios  do 
not  haemolyse,  while  many  cholera-like  vibrios  do.  The  test  can  be 
applied  in  two  ways  :  (a)  Defibrinated  rabbits'  blood  may  be  mixed 
with  melted  agar  cooled  to  45°  C.  The  mixture  is  poured  into  Petri 
dishes,  allowed  to  set,  and  when  cool  inoculated  with  the  organism 

1  As  previously  stated  (p.  175),  numerous  complements  undoubtedly 
exist,  yet  bacteria  will  absorb  both  bacteriolytic  and  haemolytic  com- 
plements. Bordet  and  Gengou  suppose  that  while  a  particular  ambo- 
ceptor has  a  maximum  avidity  for  its  homologous  complement  (which 
may  be  termed  dominant),  it  is  also  able  to  take  up  other  "  non- 
dominant  "  complements,  and  thus  bacteriolytic  amboceptor  is  able  to 
absorb  both  bacteriolytic  (dominant)  and  hsemolytic  (non-dominant) 
complements. 


FIXATION  OF  COMPLEMENT  183 

to  be  tested  in  such  a  manner  that  separate,  well-defined  colonies 
are  obtained.  After  twenty-four  hours'  incubation  at  37°  C., 
colonies  when  haemolytic  are  surrounded  with  a  clear,  well-defined 
halo  contrasting  sharply  with  the  dark  opaque  colour  of  the  agar. 
If  blood-agar  is  not  available,  a  substitute  may  be  devised  by  smear- 
ing some  sterile  human  or  rabbits'  blood  on  a  sterile  agar  plate. 
(b)  A  young  agar  culture  is  emulsified  in  4-5  c.c.  of  physiological 
salt  solution  ;  0-1  c.c.  of  this  suspension  is  mixed  in  a  tiny  test-tube 
with  O9  c.c.  of  sterile  salt  solution  and  one  drop  of  a  sterile  suspen- 
sion of  well- washed  rabbit  or  other  corpuscles.  After  twelve  to 
twenty -four  hours  haemolysis  will  be  apparent  if  the  organism  forms 
haemolysins. 

2.  Fixation  or  absorption  test.1 — A  haemolytic  serum  may  be  used 
as  a  delicate  reagent  for  complement,  and  may  thus  serve  as  a 
test  for  an  organism  or  an  immune  serum.  As  an  example  take  the 
case  of  a  supposed  cholera  vibrio.  If  an  immune  serum  (previously 
heated  to  56°  C.  so  as  to  destroy  complement) — haemolytic  for  the 
corpuscles  of  an  animal,  or  bacteriolytic  for  a  given  micro-organism, 
e.g.  cholera  vibrio — be  mixed  with  the  red  corpuscles  of  the  same 
animal,  or  with  the  cholera  vibrio,  the  corpuscles  or  the  vibrios 
respectively  absorb  the  corresponding  amboceptor  or  immune  body. 

Bordet  showed  that  if  corpuscles  or  microbes  that  have  absorbed 
the  corresponding  amboceptor  be  added  to  fresh  non-heated  comple- 
ment (e.g.  fresh  guinea-pig  serum),  the  corpuscles  or  the  microbes 
absorb  the  complement,  so  that  none  remains  free  in  the  liquid. 

But  if  fresh  guinea-pigs'  serum  be  added  to  cholera  vibrios  which 
have  not  absorbed  any  cholera  amboceptor,  the  complement  will 
not  be  absorbed  and  remains  free  in  the  liquid.  The  proof  of  this 
is  that  if  "  sensitised  "  corpuscles  (i.e.  corpuscles  which  have  taken 
up  haemolytic  amboceptor)  be  added  to  such  a  mixture,  the  globules 
are  quickly  haemolysed.  If,  on  the  other  hand,  vibrios  which  have 
already  taken  up  the  cholera  amboceptor  be  added  to  the  same 
quantity  of  fresh  serum,  the  microbe-amboceptor  complex  absorbs 
the  complement ;  and,  provided  the  amount  of  fresh  serum  is  not 
too  great,  the  complement  is  absorbed  so  completely  that  "  sensi- 
tised "  corpuscles  when  added  to  the  mixture  are  not  dissolved. 
If  vibrios  other  than  cholera  be  added  to  cholera  serum,  the  ambo- 
ceptor is  not  fixed,  the  complement  added  remains  free,  and  the 
sensitised  corpuscles  are  dissolved.  These  facts  constitute  the 
"  Bordet-Gengou  phenomenon."  The  mixture  of  an  inactivated 
haemolytic  serum  (i.e.  heated  to  56°  C.)  with  the  homologous 
corpuscles  (i.e.  those  with  which  the  haemolytic  serum  was  prepared) 

1  Often  termed  "  deviation  of  complement  "  test. 


184  A  MANUAL  OF  BACTERIOLOGY 

is  known  as  a  "  haemolytic  system."  The  following  example  illus- 
trates the  method  of  carrying  out  the  test:  The  cholera-immune 
serum  is  heated  to  56°  C.  for  half  an  hour.  An  eighteen  hours  old 
agar  culture  of  the  organism  to  be  tested  is  suspended  in  2  c.c.  of 
sterile  physiological  salt  solution.  The  complement  is  fresh  guinea- 
pig  serum  ;  a  portion  of  this  is  also  heated  to  56°  C.  (=  non-immune 
serum).  The  following  mixtures  are  prepared  in  three  small 
test-tubes  : 

Tubes  1  and  2  each  contain  0-2  c.c.  microbic  suspension  +  0-6  c.c. 

heated  immune  serum  +  0-1  c.c.  complement. 
Tube  3  contains  0-2  c.c.  microbic  suspension  +0-6  c.c.  heated 

non-immune  serum  +  0-1  c.c.  complement. 

These  are  well  shaken  to  mix  their  contents,  and  are  kept  for 
half  to  one  hour  at  37°  C.  At  the  end  of  this  time  0-1  c.c.  of  the 
following  mixture  is  added  to  tubes  1  and  3  :  two  volumes  of  heated 
(to  56°  C.  for  half  an  hour)  serum  hsemolysing  sheep's  red  corpuscles 
+  one  volume  of  washed  sheep's  corpuscles.  To  tube  2  is  added 
0-1  c.c.  of  a  mixture  of  two  volumes  of  physiological  salt  solution  + 
one  volume  of  washed  sheep's  corpuscles.  The  tubes  are  kept  for 
a  further  hour  or  so  at  37°  C.,  and  at  the  end  of  that  time  the 
occurrence  of  haemolysis  is  noted.  If  the  organism  is  homologous 
with  the  immune  serum,  the  immune  body  will  fix  the  complement 
in  tube  1  and  no  haemolysis  will  occur  ;  in  tube  3  haemolysis  will 
occur  because  the  complement  remains  free.  Tube  2  serves  as  a 
control,  and  should  show  no  haemolysis  in  three  hours  (though  if 
kept  for  eighteen  to  twenty-four  hours  haemolysis  will  occur  if  the 
organism  produces  hcemolysins,  apart  from  any  action  of  comple- 
ment). If  the  organism  is  not  homologous  with  the  immune  serum, 
haemolysis  will  occur  in  tube  1,  because  the  complement  docs  not 
become  fixed,  tubes  2  and  3  being  the  same  as  before. 

It  is  not  even  necessary  to  use  the  living  organism  ;  the  dead 
organism  or  extracts  thereof,  and,  in  cases  where  the  organism 
cannot  be  cultivated,  a  dried  and  pulverised  organ  or  an  extract 
thereof,  has  been  employed.  Certain  non-specific  substances  may 
sometimes  be  used  as  in  the  Wassermann  reaction  for  syphilis  (see 
"  Syphilis  "). 

The  haemolytic  serum  may  be  obtained  by  injecting  rabbits  with 
a  10  per  cent,  suspension  of  well-washed  sheep's  red  corpuscles. 
The  sheep's  blood  should  be  obtained  as  ascptically  as  possible 
from  the  slaughterhouse  ;  the  blood,  as  it  runs,  is  caught  in  a 
sterile  wide-mouthed  bottle  containing  a  coil  of  fine  wire  witli  which 
it  is  defibrinated  by  shaking.  The  iiuid  blood  is  then  mixed  with 


CYTOTOXINS  185 

sterile  physiological  salt  solution  (0-9-0-95  per  cent.)  and  centri- 
fuged,  and  the  deposited  corpuscles  are  again  washed  with  salt 
solution  two  or  three  times.  Three  doses  of  1  c.c.,  2  c.c.,  and  3  c.c. 
respectively  are  given  intravenously  on  successive  days,  and  after 
an  interval  of  5-7  days  the  rabbit's  serum  should  be  strongly  h«3mo- 
lytic.  Very  active  hsemolytic  sera  may  be  purchased.  The  serum 
may  be  collected  aseptically,  inactivated  by  heating  to  56°  C.  for 
half  an  hour,  and  preserved  in  sealed  ampoules.  The  activity  of 
the  hoemolytic  arnboceptor  must  be  tested  and  the  appropriate  dose 
of  it,  complement,  and  corpuscles  ascertained.  (For  manner  of 
testing,  see  "  Syphilis.") 

CYTOTOXINS.  * — Anti-sera,  analogous  to  the  hsemolysins  or  hasmo- 
toxins,  may  be  prepared  which  have  a  destructive  action  upon 
cellular  elements  ;  these  are  termed  "  cytotoxins."  If  a  rabbit  be 
injected  with  bull's  semen,  its  serum  ("  spermo toxin  ")  acquires 
the  property  of  immobilising  the  spermatozoa  of  the  bull.  The 
reaction  is  specific,  but  spermatolysis  does  not  seem  to  occur. 
Similarly,  by  injecting  ciliated  epithelium  into  the  peritoneum  of  a 
guinea-pig  an  anti-epithelial  serum,  or  "  trichotoxin,"  is  developed. 
With  liver,  kidney,  and  nerve  cells  anti-bodies  having  a  destructive 
action  upon  these  cells  are  developed  as  a  result  of  their  injection. 
Nephrotoxin,  the  serum  of  an  animal  inoculated  with  an  emulsion 
of  kidney,  when  injected  into  a  second  untreated  animal,  produces 
albuminuria  and  urajmia  with  disintegration  of  the  epithelium  of 
the  convoluted  tubules  ;  hepatotoxin,  the  serum  of  an  animal 
treated  with  emulsions  of  liver,  produces  fatty  and  inflammatory 
changes  in  the  liver  resembling  phosphorus  poisoning  ;  neurotoxin, 
the  serum  of  an  animal  treated  with  emulsions  of  nerve  tissues, 
produces  paresis,  paralysis,  depression,  convulsions,  etc.  ;  a  leuco- 
toxic  serum  obtained  by  injecting  leucocytes  agglutinates  and  dis- 
solves the  leucocytes,  and  so  on.  The  formation  and  mode  of  action 
of  these  cytotoxins  resemble  those  of  the  haemolysins.  It  was 
hoped  that  the  study  and  preparation  of  cytotoxins  would  open  up 
possibilities  in  the  way  of  treating  such  diseases  as  carcinoma  and 
sarcoma,  but  so  far  this  hope  has  not  been  realised. 

AGGLUTINATION. — If  an  animal  be  injected  with  cultures 
of  typhoid  or  cholera  bacilli,  its  serum  soon  acquires  the 
property  of  agglutinating  or  of  aggregating  into  clumps  the 
typhoid  bacilli  or  cholera  vibrios  respectively  when  mixed 
with  a  broth  culture  of  these  organisms.  The  reaction  may 
1  Sue  Uulloch,  Pracllltoiier,  May  1901,  p.  499  (Bibliog.) 


186  A  MANUAL  OF  BACTERIOLOGY 

be  observed  microscopically  in  a  hanging- drop  preparation ; 
the  organisms  first  lose  their  motility  and  soon  become 
aggregated  into  large  masses  or  clumps.  Macroscopically, 
the  reaction  may  be  followed  in  a  narrow  test-tube  into 
which  the  mixture  of  culture  and  serum  has  been  intro- 
duced ;  after  some  hours  the  micro-organisms  become 
aggregated  into  masses  so  large  as  to  form  visible  flocculi. 
The  substances  which  bring  about  this  agglutination  are 
known  as  agglutinins.  Agglutinins  seem  to  be  present 
in  small  amount  in  normal  serum ;  for  instance,  most 
normal  human  sera  up  to  a  dilution  of  1  in  2  or  1  in  4  will 
agglutinate  the  typhoid  bacillus  and  still  more  powerfully 
the  glanders  bacillus.  They  are  also  present  in  bacterial 
cultures ;  if  an  old  broth  culture  of  typhoid  be  filtered, 
the  filtrate  agglutinates  the  bacilli  in  a  fresh  broth  culture  ; 
hence  young  cultures  should  always  be  used  for  agglutina- 
tion tests.  Agglutinin  is  formed  by  the  action  of  antigen 
derived  from  the  bacterial  cell,  but  may  also  be  naturally 
present.  Agglutination  is  brought  about  by  the  action 
of  the  agglutinin  on  the  antigen ;  the  agglutinin  first 
unites  with  the  antigen,  and  this  may  occur  at  0°  C.,  and 
afterwards  exerts  its  specific  action,  which  takes  place 
only  at  higher  temperatures  and  in  the  presence  of  certain 
salts.  The  agglutinable  substance  is  known  as  aggluti- 
nogen.  Agglutinin  is  converted  into  agglutinoid  at  70°- 
75°  C.  ;  the  latter  does  not  agglutinate,  though  it  unites 
with  bacteria  and  then  prevents  the  subsequent  action  of 
agglutinin. 

The  agglutination  of  organisms  by  anti-sera,  though 
hardly  specific,  is  usually  very  special ;  given  proper 
precautions  as  to  dilution,  time-limit,  condition  of  test 
culture,  etc.,  an  anti-serum  will  generally  only  agglutinate 
the  homologous  organism  or  closely  allied  species — that  is, 
it  is  a  group  reaction.  The  anti- serum  may  agglutinate 
both  the  organism  with  which  it  has  been  prepared,  and 


AGGLUTINATION  187 

also  allied  species,  though  usually  not  to  the  same  extent ; 
anti- typhoid  serum,  for  example,  may  agglutinate  not 
only  the  typhoid  bacillus,  but  also,  though  to  a  less  degree, 
members  of  the  paratyphoid  group.  As  the  result  of 
infection  or  of  inoculation  with  an  organism,  agglutinins 
may,  however,  be  produced  which  agglutinate  not  only 
the  organism  of  the  infection,  but  also  other  organisms — 
e.g.  typhoid  serum  may  agglutinate  the  B.  coli  as  well 
as  the  B.  typhosus  and  typhus  serum  B.  typhosus  and  M. 
melitensis.  The  agglutinins  acting  on  the  infecting  organ- 
ism may  be  termed  primary  or  homologous,  those  acting 
on  other  organisms  secondary  or  heterologous.  In  a  case 
of  double  infection  each  organism  may  produce  its  own 
primary  agglutinin,  so  that  the  agglutination  of  two 
species  by  a  serum  may  be  due  to  the  presence  either  of  a 
primary  and  a  secondary  agglutinin  or  of  two  primary 
agglutinins.  Castellani,1  by  applying  the  saturation  test 
(p.  193),  found  that  an  organism  would  absorb  both  its 
primary  and  secondary  agglutinins,  but  would  not  absorb 
two  different  primary  agglutinins.  This  test,  therefore, 
would  distinguish  a  double  infection  from  a  single  one. 
Thus,  if  a  typhoid  serum  agglutinated  both  the  B.  typhosus 
and  the  B.  coli,  and  the  serum  after  saturation  with  typhoid 
culture  still  agglutinated  the  B.  coli,  this  would  point  to 
an  infection  with  the  latter  as  well  as  with  typhoid.  The 
formation  of  primary  and  secondary  agglutinins  may  be 
brought  about  as  follows  :  In  the  bacterial  cell  there  are 
several  substances,  each  of  which  forms  its  own  agglutinin. 
The  cells  of  two  bacterial  species  we  can  imagine  both 
contain  three  or  four  substances  capable  of  producing 
agglutinins,  and  it  may  happen  that  one  of  these  in  each 
species  is  the  same  and  will  produce  the  same  agglutinin — 
the  secondary  agglutinin — and,  therefore,  the  serum 
produced  by  each  bacterium  will  agglutinate  the  other. 
1  Zeitschr.  /.  Hy<j.,  xl,  1902,  p.  1. 


188  A  MANUAL  OF  BACTERIOLOGY 

The  agglutination  reaction  is  made  use  of  in  bacterio- 
logical tests  and  in  clinical  diagnosis.  The  "  Bordet- 
Durham  "  reaction  consists  in  testing  an  unknown  organism 
with  a  specific  anti- serum  prepared  by  injecting  an  animal 
with  a  known  microbe ;  if  the  organism  tested  becomes 
agglutinated,  it  is  regarded  as  being  of  the  same  species 
as  that  with  which  the  anti-serum  was  prepared.  With 
certain  precautions  the  "  Bordet-Durham "  reaction  is 
one  of  the  most  delicate  and  certain  for  the  recognition  of 
bacterial  species.  The  converse  of  this  is  the  agglutination 
reaction  proper  (frequently  termed  the  Widal  reaction), 
and  consists  in  testing  an  unknown  serum  upon  a 
known  microbe.  It  is  especially  used  in  the  diagnosis  of 
microbial  diseases ;  for  example,  in  typhoid  fever  the 
blood  of  the  typhoid  patient  powerfully  agglutinates  the 
typhoid  bacillus,  that  of  Malta  fever  the  Micrococcus 
melitensis,  that  of  bacillary  dysentery  the  dysentery 
bacillus,  etc. 

A  remarkable  phenomenon  observed  in  connection  with 
agglutination,  which  the  writer  has  particularly  noticed 
in  the  case  of  Malta  fever,  is  the  occurrence  of  what  may  be 
termed  a  zone  of  no  reaction  or  of  inhibition  with  some 
particular  dilution.  Thus,  dilutions  of  1  in  10  and  1  in  20 
may  agglutinate  strongly,  a  1  in  30,  however,  may  hardly 
agglutinate  at  all,  while  dilutions  of  1  in  40  and  upwards 
to  1  in  100  or  more  may  agglutinate  well.  A  similar 
phenomenon  has  been  observed  with  non-specific  agglu- 
tinating agents,  and  also  in  the  action  of  coagulating  agents 
on  colloid  emulsions.  Thus  orthophosphoric  acid  agglu- 
tinates a  certain  volume  of  a  suspension  of  B.  coli  when 
present  to  the  extent  of  between  118  cgrm.  and  4  cgrm., 
and  between  1-1  mgrm.  and  0-001  mgrm.,  but  not  in 
intermediate  amounts  between  40  and  1-1  mgrm. 

Anti-serum,  prepared  by  injecting  erythrocytes,  also 
agglutinates  the  red  blood- corpuscles,  and  in  certain 


THEORIES  OF  AGGLUTINATION          189 

diseases,  e.g.  pneumonia,  chromocyte  clumping  may  be  a 
marked  feature. 

Various  theories  have  been  propounded  to  account  for 
the  phenomena  of  agglutination  : 

1.  Pfeiffer  and  Emmerich  and  Loew  regarded  agglutina- 
tion as  a  vital  paralysis  of  the  bacilli  due  to  the  action  of  a 
bacteriolytic   enzyme.     Agglutination,  however,  is  not  a 
vital  phenomenon,  for  dead  bacilli  agglutinate,  and  bac- 
teriolytic enzymes  seem  to  be  destroyed  by  temperatures 
at  which  agglutinins  remain  unaffected. 

2.  Gruber,  Dineur,  and  Nicolle  supposed  that  a  glutinous 
substance,  "  glabrificin,"  is  absorbed  from  the  serum  by 
the  bacilli  causing  the  cell  membranes  or  the  flagella  to 
become  adhesive  ;  but  this  explanation  will  hardly  account 
for  the  aggregation  of  non- motile  organisms. 

3.  Paltauf  and  Duclaux  considered  that  a  precipitate 
is   produced   in   the   medium,    which   during  flocculation 
mechanically  carries  the  bacilli  with  it ;    but  there  is  no 
demonstrable  evidence  that  such  precipitation  occurs. 

4.  Bordet   separated   the   mechanism   of  agglutination 
into  two  stages — (1)  fixation  of  agglutinin,  and  (2)  aggre- 
gation.    The  fixation  of  agglutinin  by  the  organisms  he 
considers  to  be  analogous  to  the  adsorption  of  a  dye  by  a 
tissue  ;    and  once  the  agglutinin  is  fixed,  the  organisms 
obey  the  laws  of  inert  particles,  aggregation  being  caused 
by  changes  in  surface  tension,  in  the  molecular  attraction, 
between  the  organisms  and  the  surrounding  medium,  a 
view    supported    by    Craw.1     Ohno,2    however,    believes 
that  the  union  of  agglutinin  and  agglutinable  substance 
is  not  analogous  to  the  fixation  of  a  dye  by  a  tissue,  but 
that  it  is  a  chemical  combination,  as  maintained  by  Ehrlich. 

Agglutinated  bacteria  are  not  injured  by  agglutination  ; 

1  Journ.  of  Hygiene,  vol.  v,  1905,  p.  113.     See  also  Joos,  Zeitschr.  f. 
Hyg.,  xxxvi,  p.  422,  and  ibid,  xl,  p.  203. 

2  Philippine  Journ.  of  Science,  vol.  iii,  1908,  p.  47. 


190  A  MANUAL  OF  BACTERIOLOGY 

they  will,  in  fact,  grow  and  multiply  in  an  agglutinating 
serum.  The  amount  of  agglutination  does  not  bear  any 
constant  ratio  to  the  intensity  of  an  infection  ;  on  the 
whole,  if  the  patient  is  reacting  satisfactorily  to  an  infec- 
tion, the  agglutination  reaction  tends  to  be  marked  ;  if 
not,  it  may  be  feeble  or  absent.  Thus,  in  severe  typhoid 
infections  with  fatal  issue,  agglutination  may  be  absent. 
RufEer  and  Crendiropoulo *  regard  the  agglutinins  as 
being  formed  in  the  polymorphonuclear  leucocytes. 


The  Agglutination  Reaction 

A.  For  Clinical  Diagnosis  ("  Widal  "  Reaction) 

This  is  principally  made  use  of  in  typhoid  and  paratyphoid  fevers, 
Malta  fever,  and  bacillary  dysentery. 

Collection  of  blood. — Blood  is  collected  (p.  214),  preferably  in  a 
Wright's  capsule  (Fig.  35,  d,  p.  215),  or  in  a  capillary  bulbous  pipette 
(Fig.  7,  p.  52),  or  in  a  vaccine  tube.  The  ends  of  the  tube  are  sealed, 
the  dry  end  always  being  sealed  first  ;  the  blood  is  allowed  to 
coagulate  (which  may  be  hastened  by  placing  in  the  blood-heat 
incubator),  and  then  centrifuged  to  separate  the  serum,  care 
being  taken  that  the  dry  sealed  end  of  the  tube,  which  will  be 
perfectly  sealed,  is  distal  when  spinning. 

If  tubes  are  not  available,  the  blood  may  be  spotted  on  to  a 
piece  of  glass,  cover-glass,  or  slide,  glazed  paper,  tinfoil,  etc.,  and 
allowed  to  dry.  For  use,  a  drop  of  distilled  water  is  placed  on  the 
dry  blood  to  dissolve  it,  and  the  solution  used  like  serum. 

The  culture. — For  the  microscopic  test  a  young  broth  culture  is 
to  be  preferred.  A  hanging  drop  should  be  examined  to  ascertain 
that  clumps  are  absent ;  this  specimen  is  kept  as  a  control.  If 
clumps  are  present  they  may  be  removed  (in  the  case  of  typhoid) 
by  filtering  the  culture  through  filter-paper.  A  suspension  of  an 
agar  culture  may  also  be  used,  likewise  dead  cultures  :  a  broth 
culture  or  suspension  of  an  agar  one  being  heated  to  65°  C.  for  ten 
minutes  and  preserved  in  sterilised  glass  pipettes  ;  dead  cultures 
are,  however,  unsatisfactory  in  tropical  climates.  For  the  macro- 
scopic test  a  thick  suspension  of  an  agar  culture  in  salt  solution  is 
to  be  preferred,  the  suspension  being  allowed  to  sediment  for  half 

1  Brit.  Med.  Journ.,  1902,  vol.  i,  p.  821  (Bibliog.). 


THE  AGGLUTINATION  REACTION          191 

to  one  hour  before  use.  Some  strains  of  an  organism  are  better 
than  others,  and  old  laboratory  strains  are  generally  much  more 
sensitive  to  agglutination  than  recently  isolated  ones. 

Dilution  of  the  serum. — This  may  be  carried  out  in  various  ways, 
with  the  haemocytometer  pipette,  with  a  pipette  with  rubber  teat 
as  used  for  opsonin  work  (Fig.  35,  a,  p.  215),  or  with  a  platinum 
loop.  With  the  pipette  a  little  serum  is  aspirated  up  so  as  to 
occupy  1^-2  cm.  of  the  stem,  and  the  upper  limit  is  marked  with  a 
grease  pencil  or  ink.  A  bubble  of  air  is  then  admitted  so  that  an 
air-space  is  left  between  the  end  of  the  pipette  and  the  lower  end  of 
the  column  of  serum.  The  end  of  the  pipette  is  then  immersed  in 
a  watch-glass  of  salt  solution,  and  the  salt  solution  is  aspirated  up 
to  the  mark,  another  bubble  of  air  is  admitted,  and  the  process  is 
repeated  again  and  again  ;  so  that,  finally,  the  pipette  contains 
1  volume  of  serum  and  4-14  volumes  of  salt  solution,  each  volume 
being  separated  from  the  next  one  by  an  air-bubble.  The  contents 
of  the  pipette  are  then  expelled  into  a  watch-glass  and  thoroughly 
mixed,  and  further  dilution  of  this  dilution  is  performed  in  the  same 
manner.  Two  or  three  dilutions  are  usually  made — e.g.  1  in  15, 
1  in  25,  and  1  in  50.  A  platinum  loop  may  also  be  employed  as  a 
measure  ;  a  loopful  of  the  serum  is  deposited  in  a  watch-glass,  and 
by  spotting  round  it  nine  or  fourteen  loops  of  salt  solution  a  dilution 
of  1  in  10  or  1  in  15  is  prepared,  or  any  other  dilution  in  a  similar 
manner. 

The  microscopic  test. — Two  or  three  hanging-drop  slides  are 
vaselined,  and  two  or  three  cover-glasses  cleaned.  One  loopful  of 
a  dilution  of  serum  is  placed  on  each  cover-glass,  and  to  each  is 
added  a  loopful  of  the  broth  culture  of  the  organism — e.g.  typhoid — 
and  well  mixed  up,  and  the  specimens  are  mounted  as  hanging 
drops.  Starting  with  three  dilutions  of  serum — e.g.  1  in  15,  1  in  25, 
and  1  in  50 — the  dilutions  in  the  specimens  will  be  1  in  30,  1  in  50, 
and  1  in  100  respectively.  Should  only  one  dilution  of  serum  have 
been  made — e.g.  1  in  15 — if  on  each  cover-glass  one  loopful  of  this 
be  placed,  and  to  the  first  be  added  one  loopful,  to  the  second  two 
loopfuls,  and  to  the  third  three  loopfuls  of  typhoid  culture,  then  the 
final  dilutions  in  the  three  specimens  will  be  1  in  30,  1  in  45,  and 
1  in  60  respectively. 

Care  should  be  taken  that  the  hanging-drop  cultures  are  quite 
sealed  with  the  vaseline,  so  that  evaporation  is  prevented.  The 
hanging  drops  are  then  examined  microscopically,  a  £-in.  objective 
sufficing  for  typhoid.  In  the  case  of  typhoid  the  following  phenomena 
will  be  observed  :  The  motility  of  the  majority  of  the  bacilli  is 
instantaneously  or  very  quickly  arrested,  and  in  a  few  minutes  they 


192  A  MANUAL  OF  BACTERIOLOGY 

begin  to  aggregate  together  into  clumps,  and  by  the  end  of  the  half 
hour  there  will  be  very  few  isolated  bacilli  visible.  In  less  marked 
cases  the  motility  of  the  bacilli  does  not  cease  for  some  minutes, 
while  in  the  least  marked  ones  the  motility  of  the  bacilli  may  never 
be  completely  arrested,  but  they  are  always  more  or  less  sluggish 
as  compared  with  the  control  hanging  drop  made  from  the  culture, 
while  clumping  ought  to  be  quite  distinct  by  the  end  of  one  hour 
(with  a  1  in  30  to  1  in  50  dilution). 

The  central  portions  of  the  drop  should  be  examined,  not  the 
margins.  With  blood  which  has  been  dried  and  dissolved,  organisms 
may  become  entangled  in  debris,  and  must  not  be  mistaken  for 
clumps. 

In  all  cases  two  or  three  different  dilutions  should  be  made  to  exclude 
the  possibility  of  a  "  zone  of  no  reaction  "  with  some  particular  dilution 
(see  p.  188). 

Macroscopic,  or  sedimentation  method. — The  serum,  having  been 
diluted  by  means  of  a  pipette  with  four  times  its  volume  of  salt 
solution,  is  mixed  with  five  to  twenty  times  its  volume  of  culture 
suspension  containing  plenty  of  micro-organisms  in  the  same  manner 
as  described  in  the  previous  section.  The  mixture  is  sucked  up  into 
a  fine,  but  not  capillary,  bore  tube.  This  is  sealed  at  the  lower  end 
and  allowed  to  stand  in  the  upright  position  for  eight  to  twenty-four 
hours  at  20°  C.,  or  six  hours  at  37°  C.  ;  the  reaction  is  often  distinct 
within  an  hour  at  37°  C.  When  the  reaction  is  posit  i  ve  t  he  organisms 
become  agglutinated,  and  form  flocculi,  which  are  easily  seen  wilh 
the  naked  eye  or  with  a  hand-lens  and  stick  to  the  sides  or  sink  to 
the  bottom  of  the  tube.  The  dilution  usually  employed  is  1  in  30 
to  1  in  50.  Whole  blood  is  not  suitable  for  the  sedimentation  test  ; 
clear  serum  should  always  be  used.  It  is  well  to  set  up  at  the  same 
time  a  control  tube  with  saline  solution,  or,  preferably,  with  normal 
serum. 

If  sufficient  serum  is  available  the  mixture  may  be  put  up  in 
little  test-tubes,  such  as  the  inner  tubes  of  Durham's  culture-tubes 
(p.  83). 

B.  For  the  Recognition  of  Bacterial  Species 

1.  Bordet-Durham  reaction. — This  is  carried  out  in  much  the 
same  manner  as  for  clinical  diagnosis,  but  an  immune  serum  of 
high  agglutinating  value  or  high  "  titre  "  (at  least  1  :  1000)  is 
required,  and  the  serum  from  a  patient  is  not  applicable.  The 
immune  serum  may  be  obtained  from  a  horse  or  other  animal 
immunised  with  killed  cultures  (and  living  also  if  a  high  titre  is 
required).  In  the  laboratory  the  serum  may  be  prepared  by  giving 


THE  MEIOSTAGMIN  KEACTION  193 

a  rabbit  three  to  five  intravenous  injections  at  intervals  of  seven 
days  of  killed  culture  of  a  virulent  strain  of  the  organism,  e.g. 
typhoid  or  cholera.  The  culture  is  killed  by  heating  to  60°-65°  C. 
for  half  an  hour,  and  the  dose  is  increased  from  one  loop  to  ten 
loops  of  an  agar  culture.  Seven  days  after  the  last  dose  the  animal 
is  bled  from  an  ear  vein,  and  the  serum  obtained.  The  agglutinating 
value  of  the  serum  must  be  determined,  and  controls  should  always 
be  put  up  with  normal  serum  of  an  animal  of  the  same  species  as 
that  from  which  the  immune  serum  has  been  obtained.  A  series  of 
dilutions  of  both  sera  is  made  with  salt  solution  and  a  twenty-four 
hour  agar  culture  of  the  organism  to  be  tested  used.  Both  the 
macroscopic  and  microscopic  methods  should  be  employed.  The 
dilutions  may  be  made  with  a  1  c.c.  pipette  graduated  in  hundredths, 
with  the  haemocytometer  pipettes,  or  by  the  method  used  clinically. 

2.  Saturation  test. — Castellani  noticed  that  a  suspension  of  a 
microbe  added  to  the  homologous  agglutinating  serum  absorbs 
most,  if  not  all,  the  specific  agglutinin,  whereas  an  organism  not 
homo]ogous  with  the  serum  absorbs  little  or  only  a  portion  of  the 
agglutinin.  The  test  may  be  carried  out  as  follows  : 

Ten  loopfuls  of  a  young  agar  culture  of  the  organism  to  be  tested 
are  mixed  with  10  c.c.  of  a  5  per  cent,  solution  of  a  highly  aggluti- 
nating serum.  After  incubating  for  two  or  three  hours,  the  mix- 
ture is  centrifuged,  the  clear  supernatant  fluid  decanted,  and 
the  agglutinating  power  of  the  decanted  liquid  is  then  tested  on  the 
organism  with  which  the  serum  was  prepared.  If  the  organism 
tested  is  homologous  with  the  organism  with  which  the  agglutinat- 
ing serum  was  prepared,  the  decanted  fluid  will  have  lost  most, 
or  a  considerable  proportion,  of  its  agglutinating  power  for  the 
latter. 

THE  MEIOSTAGMIN  REACTION. — Ascoli  has  found  that  if  an 
immune  serum  be  mixed  with  an  alcoholic  extract  of  the  homologous 
antigen  and  the  mixture  incubated  at  37°  C.  for  two  hours  the 
surface  tension  is  reduced  ;  if  the  serum  and  antigen  extract  are 
not  homologous  the  surface  tension  is  unaltered.  For  example,  in 
the  case  of  typhoid  the  following  is  the  procedure.  An  alcoholic 
extract  of  typhoid  bacilli  is  prepared  ;  this  is  diluted  with  saline 
solution  to  1-1000 — 1-1,000,000.  The  typhoid  serum  is  similarly 
diluted,  1-10.  To  9  c.c.  of  the  diluted  serum  1  c.c.  of  the  diluted 
antigen  extract  is  added.  By  means  of  some  form  of  viscosimeter 
or  stalagmometer  the  number  of  drops  yielded  by  a  given  volume 
of  the  mixture  is  ascertained,  immediately  after  the  mixture  is 
made  and  after  the  mixture  has  been  incubated  at  37°  C.  for  two 
hours.  If  the  surface  tension  has  been  reduced,  the  number  of 

13 


194  A  MANUAL  OF  BACTERIOLOGY 

drops  counted  in  the  second  determination  will  be  greater  than  in 
the  first.1 

ANTI-FERMENTS.  2 — By  the  injection  of  rennin  or  other  enzyme 
the  blood-serum  of  the  treated  animal  acquires  the  property  of 
neutralising  the  action  of  the  enzyme  with  which  the  inoculation 
has  been  performed.  Thus  if  rennin  and  anti -rennin  (the  serum  of 
an  animal  injected  with  rennin)  be  mixed  with  milk  no  curdling 
takes  place.  Similarly,  the  serum  of  an  animal  inoculated  with 
pancreatin  inhibits  the  action  of  this  ferment,  and  if  coagulated 
egg-albumen,  pancreatin,  and  anti -pancreatin  be  mixed,  the  egg- 
albumen  undergoes  no  digestion. 

PnECiPiTiNS.3 — Kraus  was  the  first  to  demonstrate  the  presence 
of  specific  precipitins  in  blood  by  adding  typhoid,  cholera,  and  plague 
anti-sera  to  filtrates  of  the  cultures  of  the  corresponding  microbes. 
If  to  such  a  filtrate  in  a  test-tube  a  little  of  the  corresponding 
anti -serum  be  added  by  running  in  carefully,  so  that  it  forms  a  layer 
at  the  bottom,  an  opalescent  ring  makes  its  appearance  at  the  line 
of  junction  of  the  two  fluids.  So  also  if  an  animal  be  injected  with 
milk,  its  serum,  when  added  to  milk  of  the  same  kind  as  that  with 
which  it  has  been  injected,  causes  precipitation  of  the  casein.  This 
reaction  is  specific,  and  it  is  thus  possible  to  distinguish  various 
milks  from  one  another.  Similarly,  anti-sera  which  produce  pre- 
cipitates, each  with  the  homologous  substance,  are  obtained  by 
the  injection  of  peptone,  of  egg-albumen,  blood-serum,  and  other 
proteins.  The  latter  reaction  has  an  important  medico-legal 
application,  for  by  means  of  it  the  blood  and  flesh  of  different 
species  of  animals  can  be  distinguished.  Thus  the  presence  of 
horseflesh  in  sausages  can  be  detected.  The  method  employed  is 
to  inject  a  rabbit  intraperitoneally  with  four  to  six  injections  of 
defibrinated  blood  or  of  blood-serum  (or  with  a  solution  of  the 
particular  substance,  e.g.  horseflesh),  commencing  with  about  5  c.c. 
and  increasing  to  10  c.c.  at  intervals  of  a  few  days.  After  treat- 
ment the  animal  is  bled  from  an  ear  vein,  and  the  serum  is  obtained. 
The  blood  to  be  tested  may  be  dried  on  filter-paper,  pieces  are  then 
cut  up,  a  solution  is  made  in  1-6  per  cent,  sodium  chloride  solution, 
and  to  this  the  specific  serum  is  added.  Tested  in  this  way  human 
blood  anti-serum  reacts — i.e.  forms  a  precipitate — markedly  with 

1  Ascoli  and  Izar,  Munch,  med.  Woch.,  Ivii,  1910,  pp.  62,  182,  403. 

2  See  Dean,  Trans.  Path.  Soc.  Lond.,  vol.  lii,  1901,  Part  2,  p.  127. 

3  See  Nuttall,  Journ.  of  Hyg.,  vol.  i,  1901,  p.  367  (Bibliog.),  also  Brit. 
Med.  Journ.,   1902,  vol.  i,  p.   825  ;    Welsh  and  Chapman,  Journ.   of 
Hygiene,  vol.  x,  1910,  p.  177  ;  ibid.  Australasian  Med,  Gazette,  December 
12.  1908  (hydatid  disease). 


IMMUNITY  195 

human  blood,  less  so  with  ape's  blood,  not  at  all  with  other  blood  ; 
ox  blood  anti-serum  reacts  with  ox  blood,  less  so  with  sheep,  feebly 
with  horse,  hardly  at  all  with  dog.  Mixtures  of  bloods  may  also 
be  tested.  Precipitins  are  also  formed  naturally  in  vivo.  Thus 
the  serum  of  a  patient  the  subject  of  hydatid  disease  gives  a  precipi- 
tate with  hydatid  fluid,  and  the  reaction  may  be  used  diagnostically. 
The  production  of  the  anti-body  seems  to  be  due  to  the  globulin 
constituent  of  the  injected  serum. 

It  will  thus  be  seen  that  the  anti-bodies  which  result 
from  the  injection  into  an  animal  of  different  substances 
are  extremely  numerous  and  have  varied  properties,  their 
most  notable  characteristics  being  their  extreme  specificity 
and  the  extraordinary  delicacy  of  the  interactions  produced 
by  them.  It  is  important  to  note  that  these  anti-bodies 
are  produced  only  as  the  result  of  inoculation  with  complex 
compounds  allied  to  the  proteins.  The  tolerance  estab- 
lished by  the  ingestion  or  inoculation  of  simpler  com- 
pounds, such  as  arsenious  acid  and  morphine,  is  of  a  different 
nature,  and  is  not  coincident  with  the  development  of 
anti-bodies.  According  to  Ehrlich,  the  latter  kind  of 
tolerance  may  be  due  to  the  exhaustion  or  using  up  of 
certain  receptors  ("  chemo-receptors  ")  of  the  protoplasm 
(see  p.  206). 

Immunity * 

No  fact  in  biology  is  more  striking  than  the  differences 
in  susceptibility  to  infection  exhibited  by  different  races 
and  different  animals.  For  example,  the  natives  in  many 
parts  of  the  world  are  comparatively  insusceptible  to  yellow 
and  typhoid  fevers  and  malaria,  the  dog  and  goat  are  rarely 
affected  with  tuberculosis,  and  tetanus  is  never  met  with 
in  the  fowl ;  and  to  come  nearer  home,  while  some  indi- 
viduals are  lucky  enough  to  escape  most  of  the  commoner 

1  See  Metchnikoff,  Immunity  in  Infective  Diseases,  1905.  Also  Brit. 
Med.  Journ.,  1902,  vol.  i,  p.  784  ;  1904,  vol.  ii,  pp.  557-582  ;  and  1907, 
vol.  ii,  pp.  1409-1425  ;  Journ.  of  Hygiene,  vol.  ii,  1902  ;  Emery,  Im- 
munity and  Specific  Therapy,  1909. 


196  A  MANUAL  OF  BACTERIOLOGY 

infectious  fevers,  others  seem  to  contract  them  on  every 
possible  occasion,  and  to  suffer  from  all  the  ills  that  flesh 
is  heir  to.  These  instances  show  that  there  is  often  a 
natural  insusceptibility  to  infective  disease,  or  a  natural 
immunity,  as  it  is  termed.  This  may  be  complete  or 
partial,  or  it  may  appertain  only  to  a  race — "  racial 
immunity  "  ;  or,  varying  in  different  individuals  and  at 
different  ages,  it  constitutes  "  individual  immunity,"  as 
in  the  case  of  diphtheria  and  scarlatina,  which  become 
more  and  more  rare  as  age  advances. 

Still  more  striking,  perhaps,  is  the  fact  that  an  insus- 
ceptibility may  be  acquired  after  an  attack  of  infective 
disease  or  be  conferred  in  certain  instances  by  inoculation. 
Thus  second  attacks  of  smallpox  and  scarlatina  are  rare, 
inoculated  smallpox  and  vaccinia  protect  against  variola, 
and  bacterial  vaccines  confer  considerable  protection. 

With  regard  to  the  immunity  of  native  races  to  certain 
diseases,  this  is  probably  due  to  natural  selection  and 
heredity  ;  during  long  periods  of  time,  the  individuals 
being  all  exposed  to  the  same  risks,  the  susceptible  ones 
are  weeded  out,  while  the  survivors  transmit  their  insus- 
ceptibility to  their  descendants  ;  but  this,  of  course,  does 
not  explain  the  reason  for  the  relatively  greater  immunity 
of  the  insusceptible  individuals.  Immunity  is  generally 
not  absolute  either  to  infection  or  intoxication  ;  that  is, 
infection  can  usually  be  induced  under  certain  conditions. 
Thus  fowls,  which  are  highly  refractory  to  tetanus  and 
tolerate  considerable  doses  of  tetanus  toxin  with  impunity, 
can  be  tetanised  with  large  doses  of  an  active  toxin  ;  white 
rats,  which  are  insusceptible  to  anthrax,  become  susceptible 
after  fatigue,  or  when  fed  on  an  exclusively  vegetable  diet. 
Immunity  is  therefore  either  (1)  natural,  or  (2)  acquired, 
and  it  is  evinced  against  either  (a)  toxins,  or  (6)  micro- 
organisms, and  these  different  phases  must  be  con- 
sidered. 


IMMUNITY  197 

1.  Natural  immunity  against  toxins. — There  are  various 
non-specific  reactions  in  the  body  by  which  toxins  may 
be  eliminated  or  destroyed.  Thus  the  dilatation  of  the 
vessels  and  the  acceleration  of  the  blood-stream  which 
take  place  in  an  inflamed  area  dilute  and  eliminate  the 
toxin,  and  the  proteolytic  enzymes  produced  by  the 
organisms  and  as  a  result  of  tissue  disintegration  may 
have  a  destructive  action  on  the  toxins.  Oxidation, 
hydration  and  dehydration,  and  various  analytic  and 
synthetic  processes  which  go  on  in  the  body,  and  particu- 
larly in  the  liver,  are  other  agencies  whereby  toxins  may  be 
destroyed.  These  non-specific  processes  by  which  toxin 
is  destroyed  or  eliminated,  though  of  the  greatest  impor- 
tance, can  probably  deal  with  only  small  amounts  of  toxin  ; 
if  large  amounts  are  present,  specific  reactions  have  to  be 
evoked. 

Another  cause  of  natural  immunity  to  toxins  may  be 
the  absence  of  suitable  receptors  for  the  toxin.  As  already 
stated  (p.  153),  in  order  that  a  bacterial  toxin  or  endotoxin 
may  produce  intoxication,  it  must  become  anchored  to 
the  cells  by  its  haptophore  group,  and  that  this  may  occur 
the  cell  molecules  must  possess  atomic  groups  or  side- 
chains  ("  receptor  groups  ")  which  have  a  special  affinity 
for  the  haptophore  groups  of  the  toxin.  Should  these  be 
wanting  the  toxin  cannot  become  anchored  to  the  cells, 
its  toxophore  groups  cannot  exert  their  influence,  and 
natural  immunity  is  the  result. 

This  has  been  proved  to  be  the  case  in  several  instances. 
Thus  in  the  lizard  and  turtle,  if  tetanus  toxin  be  injected 
no  effect  is  produced,  but  the  toxin  is  not  eliminated  and 
remains  in  the  body  for  months,  as  may  be  proved  by 
withdrawing  a  little  of  the  blood  and  injecting  it  into  a 
mouse  ;  the  animal  dies  of  tetanus. 

In  other  instances,  for  some  reason  or  other,  the  cells 
of  the  animal  are  insusceptible  to  the  toxophore  group  of 


198  A  MANUAL  OF  BACTERIOLOGY 

the  toxin.  Thus,  if  an  alligator  be  injected  with  tetanus 
toxin,  no  effect  is  produced,  but  the  toxin  rapidly  disappears 
from  the  blood.  If  the  animal  be  kept  at  ordinary  tem- 
perature (20°  C.),  although  the  toxin  disappears,  antitoxin 
is  not  formed,  but  if  it  is  kept  at  30°-37°  C.  antitoxin  is 
rapidly  produced.  The  two  experiments  together  suggest 
that  the  toxin  is  fixed  by  the  cells,  but  has  no  effect  upon 
them  ;  if  the.  toxin  were  not  fixed,  it  would  be  possible  to 
detect  it,  and  presumably  it  would  not  produce  antitoxin. 
2.  Natural  immunity  against  micro-organisms. — A  number 
of  factors  are  doubtless  concerned  in  preserving  the  body 
from  invasion  by  micro-organisms,  and  while  non-specific 
reactions  may  suffice  when  the  number  of  organisms  is 
small,  specific  reactions  have  to  be  evoked  if  the  number 
of  organisms  is  large.  The  unbroken  surfaces  of  the 
body  have  a  considerable  protective  action  in  preventing 
the  entrance  of  micro-organisms.  The  flushing-out  action 
of  accelerated  circulation  will  exert  some  action  in  elimina- 
ting organisms  from  a  localised  focus  of  infection  just  as 
it  does  with  toxins.  The  body  temperature  may  be  of 
some  importance,  and  the  febrile  condition  so  generally 
induced  by  infection  is  probably  to  some  extent  protective 
and  curative.  Thus  frogs,  fish,  and  chickens  are  naturally 
immune  to  anthrax.  In  the  one  case  the  body  tempera- 
ture is  low,  18°  C.  or  thereabouts  ;  in  the  other  it  is  high, 
40°  to  41°  C.,  and  this  may  influence  the  growth  of  the 
anthrax  bacillus,  preventing  the  full  and  rapid  development 
which  may  be  necessary  for  the  production  of  the  disease. 
That  such  is  the  case  would  seem  to  be  shown  by  experi- 
ments in  which  when  the  temperature  of  the  medium  is 
raised  or  lowered,  infection  takes  place  ;  frogs  and  fish 
kept  in  water  raised  to  a  temperature  of  35°  C.,  and  chicken 
refrigerated  so  as  to  reduce  their  temperature,  all  perish 
from  anthrax  after  inoculation.  It  is  clear,  however,  that 
this  is  not  necessarily  the  only  factor,  for  sparrows,  which 


IMMUNITY  199 

have  a  temperature  as  high  as  that  of  the  chicken,  can 
be  infected  with  anthrax  without  refrigerating.  Behring 
would  ascribe  the  immunity  of  white  rats  to  anthrax  to 
the  high  alkalinity  of  their  blood,  and  claims  to  have  shown 
experimentally  that  a  vegetable  diet  reduces  this,  and 
fatigue  is  said  to  act  similarly. 

In  some  cases  the  animal,  after  invasion  by  the  organism, 
becomes  gradually  tolerant  to  its  presence  (immunitas  non 
sterilisans).  This  is  particularly  the  case  in  protozoan 
infections,  e.g.  piroplasmosis.  The  animal,  after  a  period 
of  ill-health,  gradually  recovers,  though  the  organisms 
may  still  be  present,  as  can  be  demonstrated  by  injecting 
some  of  its  blood  into  a  susceptible  animal.  Conceivably 
the  receptors  necessary  for  the  intoxication  become 
gradually  used  up,  and  when  this  state  is  attained  the 
animal  becomes  insusceptible. 

The  blood,  lymph,  and  other  fluid  and  tissue  juices 
undoubtedly  exert  a  more  or  less  germicidal  action  on 
bacteria  experimentally  in  vitro,  and  to  some  extent 
probably  also  in  the  body.  But  in  this  respect  there  is 
often  a  marked  difference  between  the  circulating  blood 
and  the  blood  in  vitro. 

Lewis  and  Cunningham  (1872),  Traube  and  Gscheidlen 
(1874),  Fodor  (1877),  and  Wysokowicz  showed  that  bac- 
teria injected  into  the  circulation  rapidly  disappear,  and 
were  inclined  to  attribute  this  result  to  the  bactericidal 
properties  of  the  blood.  In  the  main,  however,  this  dis- 
appearance is  due  to  lodgment  in  the  capillaries,  phago- 
cytosis, and  excretion  by  the  excretory  glands. 

Halliburton  prepared  from  the  lymphatic  glands  a 
protein,  cell-globulin  /3  (really  a  nucleo-protein).  Hankin 
found  that  this  substance  had  marked  germicidal  properties, 
and  concluded  that  it  was  probably  the  germicidal  con- 
stituent of  the  blood-serum.  Bitter,  who  repeated  Hankin's 
experiments,  failed,  however,  to  confirm  them.  To  the 


200  A  MANUAL  OF  BACTEKIOLOGY 

germicidal  constituents  of  the  cells  and  body  fluids  Buchner 
gave  the  name  "  alexins." 

Grohmann  performed  the  first  experiments  with  extra- 
vascular  blood.  He  found  that  anthrax  bacilli,  after  being 
kept  in  plasma,  became  less  virulent.  Fodor,  adding 
anthrax  bacilli  to  blood  and  plating  at  intervals,  found 
there  was  a  progressive  diminution  in  the  number  of 
organisms. 

Nuttall,  in  1888,  used  the  defibrinated  blood  of  several 
animals,  rabbits,  mice,  pigeons,  sheep,  and  found  that  it 
destroyed  the  B.  anthracis,  B.  subtilis,  B.  megaterium,  and 
M.  pyogenes  var.  aureus.  He  confirmed  Fodor's  results, 
which  also  showed  that  after  a  while  the  blood  loses  its 
germicidal  properties  and  becomes  a  suitable  culture 
medium.  The  blood  or  serum  similarly  loses  its  bactericidal 
properties  on  heating,  and  serum  that  has  once  been  used 
loses  its  bactericidal  properties.  Nissen  continued  this 
work,  and  also  found  that  fresh  serum  is  germicidal  for  a 
variety  of  organisms. 

In  1890,  Buchner  with  Voit,  Sittmann,  and  Orthen- 
berger  came  to  the  conclusion  that  the  germicidal  action 
of  cell-free  serum  is  due  to  the  protein  constituents. 

Christmas  prepared  a  germicidal  substance  from  the 
spleen,  and  Bitter,  who  examined  the  method,  in  the  main 
confirmed  Christmas. 

Behring  and  Nissen,  however,  found  that  the  serum 
of  the  white  rat,  dog,  and  rabbit  destroys  the  Bacillus 
anthracis,  but  serum  from  the  mouse,  sheep,  guinea-pig, 
chicken,  pigeon,  and  frog  has  no  action.  Thus,  while  the 
rabbit  is  highly  susceptible  to  anthrax,  its  serum  is  germi- 
cidal ;  the  chicken,  on  the  other  hand,  is  immune  to 
anthrax,  but  its  serum  is  inactive.  Hence  there  is  a 
considerable  difference  between  the  action  of  circulating 
and  of  extra-vascular  blood. 

Vaughan,  Novy  and  McClintock,  in  a  series  of  papers, 


IMMUNITY  201 

ascribed  powerful  bactericidal  properties  to  the  nucleins, 
and  surmised  that  in  serum  the  nucleins  set  free  by  the 
disintegration  of  leucocytes  and  other  cells  are  the  germi- 
cidal  agents.  Forrest  and  the  writer 1  found,  however, 
that  all  the  germicidal  properties  ascribed  by  Vaughan 
to  the  nucleins  are  probably  due  to  the  weak  alkali  in 
which  the  nucleins  were  dissolved,  and  came  to  the  con- 
clusion that  Vaughan's  results  are  at  least  not  proven. 

Gengou  also  found  that  the  plasma  collected  in  vaselined 
tubes  is  often  almost  devoid  of  bactericidal  power,  whilst 
the  corresponding  serum  may  be  capable  of  destroying 
large  numbers  of  micro-organisms. 

We  therefore  see  that  while  the  blood,  lymph,  and 
other  fluids  and  tissue  juices  undoubtedly  exert  more  or 
less  germicidal  action  on  bacteria  experimentally  in  vitro, 
there  is  often  a  marked  difference  in  this  respect  between 
the  circulating  blood  and  the  blood  in  vitro  and  it  may  be 
doubted  if  this  factor  is  of  great  importance  in  the  produc- 
tion of  natural  immunity.  At  the  same  time,  it  is  to  be 
noted  that  directly  infection  has  started  more  or  less  cel- 
lular disintegration  and  serous  exudation  occur,  and  thus 
the  germicidal  action  of  the  body  fluids  and  tissues  may 
be  exerted  in  vivo,  though  such  substances  may  act  rather 
by  stimulating  the  leucocytes  or  by  rendering  the  bacteria 
more  phagocytosable,  as  will  be  referred  to  later  (p.  209). 
Thus  Kanthack  and  Hardy  found  that  the  coarsely 
granular  oxyphile  leucocytes  in  the  frog  are  first  attracted 
to  the  site  of  a  bacterial  invasion,  there  discharge  their 
oxyphile  granules,  the  bacteria  then  show  signs  of  degenera- 
tion, and  polymorphonuclear  leucocytes  and  other  "  phago- 
cytic "  cells  now  approach  and  ingest  the  degenerate 
bacteria.  The  observations,  however,  do  not  seem  to  have 
been  confirmed.  Wooldridge  also  protected  animals  from 
anthrax  by  injections  of  "  tissue  fibrinogen "  (nucleo- 

1  Journ.  Roy.  Army  Hed.  Corps. 


202  A  MANUAL  OF  BACTERIOLOGY 

protein).  For  some  micro-organisms  a  bacteriolytic 
mechanism  exists,  the  amboceptor- complement  complex, 
whereby  they  may  be  digested  and  got  rid  of.  Thus 
normal  serum  has  a  marked  bacteriolytic  action  on  B. 
typhosus  and  B.  coli.  In  many  cases,  however,  e.g.  for 
staphylococci,  such  a  bacteriolytic  mechanism  does  not 
naturally  exist,  but  may  be  evoked  as  a  result  of  infection. 

The  hypothesis  which  ascribes  immunity  to  the  germi- 
cidal  and  bacteriolytic  action  of  substances  in  the  fluids 
of  the  body  has  been  termed  the  "  humoral  theory." 

Another  important  theory  of  immunity  is  the  doctrine 
of  phagocytosis,  so  ably  supported  by  MetchnikofL  This 
is  the  "  cellular  "  theory  of  immunity.  It  has  as  its  basis 
the  following  fundamental  facts  :  Firstly,  the  leucocytes 
in  the  circulating  blood  ingest  and  destroy  any  foreign 
particles  present  therein ;  secondly,  an  injury  to  the 
tissues  is  immediately  followed  by  an  inflammatory  reac- 
tion, in  which  the  leucocytes  emigrate  from  the  vessels 
and  congregate  at  the  injured  spot.  Similarly,  in  many 
instances  the  leucocytes  rapidly  congregate  at  the  seat  of 
a  bacterial  infection,  and  approach  and  engulf  the  bacteria 
in  the  same  manner  as  they  do  other  foreign  particles,  and 
so  rid  the  body  of  the  unwelcome  guests  (Plate  I.,  a  and  b). 

The  migration  of  the  leucocytes  towards  the  scene  of 
action  is  explained  by  MetcrmikofT  on  the  hypothesis  that 
the  chemical  substances  elaborated  by  the  bacteria  attract 
the  latter  and  exert  what  he  termed  "  positive  chemo- 
taxis."  In  this  case  the  bacteria  are  removed  by  the 
leucocytes,  and  general  infection  and  death  do  not  occur. 
But,  unfortunately,  in  other  cases  the  bacterial  chemical 
products  repel,  or  perhaps  it  is  more  correct  to  say  do  not 
attract,  the  leucocytes,  and  "  negative  chemotaxis  "  occurs, 
so  that  the  bacteria  are  free  to  grow  and  multiply,  and 
general  infection  ensues.  Positive  and  negative  chemo- 
taxis can  be  shown  to  occur  by  a  simple  experiment.  If 


IMMUNITY  203 

a  fine  capillary  tube  containing  some  peptone  solution  be 
introduced  into  a  suspension  of  bacilli,  e.g.  B.  fluorescens 
liquefaciens,  under  a  cover- glass,  and  watched  microscopi- 
cally, the  bacilli  will  be  attracted  to  the  tube  and  soon 
invade  its  lumen.  If,  however,  a  weak  acid  be  substituted 
for  the  peptone  water,  the  bacilli  will  be  repelled.  The 
process  by  which  the  bacteria  are  ingested  by  the  leucocytes 
can  be  similarly  watched.  The  leucocytes  which  act  in 
this  manner  are  termed  phagocytes,  and  they  are  of  two 
classes — the  macrophages,  the  large  mononuclear  leuco- 
cytes, and  the  smaller  microphages,  or  polymorphonuclear 
leucocytes.  Certain  of  the  tissue  cells  and  endothelial 
cells  also  possess  phagocytic  properties.  The  importance 
of  phagocytosis  is  also  shown  by  the  fact  that,  while  in 
ordinary  susceptible  rabbits  infection  with  anthrax  is 
followed  by  a  feeble  phagocytosis  and  the  animals  succumb, 
in  rabbits  vaccinated  against  anthrax  phagocytosis  is  very 
active.  Moreover,  in  an  animal  refractory  to  anthrax, 
such  as  the  frog,  anthrax  bacilli  grow  and  multiply  if  they 
be  enclosed  in  paper  or  collodion  sacs,  so  as  to  prevent  the 
access  of  the  phagocytes. 

Phagocytosis,  in  vitro,  and  probably  also  in  the  normal 
body,  is  extraordinarily  active,  so  that  it  might  be  expected 
always  to  be  sufficient  to  deal  with  any  number  of  bacteria 
that  might  be  introduced.  If,  however,  the  bacteria  be 
virulent,  negative  chemotaxis  will  occur.  Moreover,  the 
presence  of  substances  which  render  the  bacteria  phago- 
cytosable,  "  opsonins,"  is  necessary,  and  it  seems  likely 
that  the  amount  of  opsonin  becomes  diminished  in  infection 
(see  p.  211). 

Metchnikoff  admits  that  the  destruction  of  bacteria  in 
phagocytosis  is  brought  about  by  chemical  bacteriolytic 
substances,  which  he  terms  "  cytases,"  and  which  he 
regards  as  being  derived  from  the  leucocytes,  and  as 
identical  with  the  alexins.  He  believes  that  there  are  two 


204  A  MANUAL  OF  BACTEEIOLOGY 

kinds  of  cytases,  one  "  macrocytase,"  obtainable  from 
tissues,  such  as  the  spleen  and  lymph- glands,  rich  in 
macrophages,  which  acts  specially  on  elements  of  animal 
origin,  the  other  "  microcytase,"  derived  from  the  micro- 
phages,  and  which  acts  principally  on  micro-organisms. 
He  considers  the  alexic  action  to  be  of  the  nature  of  a 
digestive  process  (but  this  is  doubtful),  and  as  regards  the 
complex  nature  of  a  cytolytic  serum,  which  contains  ambo- 
ceptor  and  complement,  believes  that  the  amboceptor  is 
formed  within  the  macrophages  in  intra-cellular  digestion, 
and  that  a  portion  of  it  escapes  from  them  into  the  serum. 
All  the  facts  point  to  the  leucocytes  and  leucocytic  tissues 
being  the  great  defensive  mechanisms  against  parasitic 
invasion,  either  by  the  production  of  alexins,  or  of  bacterio- 
lysins,  or  by  phagocytosis,  or  probably  by  a  combination 
of  these  (the  "  cellulo-humoral  "  hypothesis  of  immunity). 
It  is  probable  that  the  greater  part  of  phagocytosis  takes 
place  in  the  spleen.  This  organ  acts  as  a  sort  of  filter, 
and  phagocytosis  may  be  active  in  it  when  none  can  be 
discerned  in  the  blood.  Phagocytosis  is  also  active  in  the 
bone-marrow. 

Experiments  by  Tizzoni  and  Cattani  seemed  to  show 
that  rabbits  could  not  be  rendered  refractory  to  tetanus 
by  injection  of  tetanus  antitoxin  after  extirpation  of  the 
spleen  ;  and  although  Benario  and  other  observers  have 
not  confirmed  this,  the  manner  in  which  the  spleen  is 
attacked  in  such  diseases  as  tuberculosis,  plague,  etc., 
points  to  this  conclusion.  The  discordant  results  obtained 
after  splenectomy  may  be  due  to  the  rapid  regeneration 
of  spleen  tissue,  and  to  other  structures,  such  as  the 
hsemolymph  glands,  taking  on  its  functions  after  ablation. 

Although  small  amounts  of  antitoxin  may  occasionally 
be  met  with  in  the  normal  animal  (e.g.  diphtheria  anti- 
toxin in  man  and  in  the  horse,  see  pp.  153  and  274),  this 
substance  plays  little  or  no  part  in  natural  immunity 


IMMUNITY  205 

against  either  toxin  or  micro-organism.  Thus  the  blood- 
serum  of  the  fowl,  which  is  highly  refractory  to  tetanus 
does  not  exert  the  slightest  antitoxic  or  neutralising  action 
on  tetanus  toxin. 

3.  Acquired  immunity. — Acquired  immunity  may  be 
induced  in  several  ways  : 

(1)  By  an  attack  of  the  disease  ending  in  recovery. 

(2)  By  vaccinating  with  a  modified  and  less  virulent 
form  of  the  living  infective  agent  (Pasteur's  method). 

(3)  By    treatment    with    sterilised    cultures,    or    with 
bacteria-free  toxins. 

(4)  Occasionally  by  treatment  with  sterilised  cultures 
or  toxins   of  a   different  species.     Thus,   B.   pyocyaneus 
protects  from  anthrax  (p.  238),  and  Klein1  showed  that 
an   injection   of    one  of    the   six  following    organisms — 
(1)  Koch's  comma,  (2)  Finkler-Prior's  comma,  (3)  B.  coli, 
(4)  Proteus  vulgaris,  (5)  B.  prodigiosus,  (6)  B.  typhosus — 
will  protect  an  animal  against  any  one  of  the  remaining 
five.     He  therefore  concluded  that  there  is  an  immunising 
agent  common  to  all  these  six  organisms,  and  that  this 
substance  is  intra- cellular  and  a  constituent  of  the  bacterial 
cells  themselves.     In  this  case,  however,  the  immunity  is 
probably  one  against  certain  bacterial  proteins  and  not 
against  the  specific  endotoxins  of  the  organisms. 

(5)  By  injection  of  the  blood-serum  derived  from  an 
animal  treated  or  immunised  by  method  (3) — that  is  to  say, 
antitoxins    or   other   anti-bodies    (e.g.    amboceptors)    are 
introduced. 

The  immunity  acquired  by  methods  (l)-(4)  is  known  as 
"  active  immunity,"  because  the  animal's  cells  and  tissues 
are  altered  by  the  process,  so  that  they  are  no  longer 
susceptible  to  the  microbe  or  its  toxin.  The  immunity 
conveyed  by  method  (5) — the  injection  of  an  immune 
serum,  is  known  as  "  passive  immunity,"  because  the 

1  Trans.  Path.  Soc.  Lond.,  1893,  p.  220. 


206  A  MANUAL  OF  BACTERIOLOGY 

immunity  lasts  only  so  long  as  the  anti-bodies  remain  ; 
there  is  no  active  participation  of  the  animal's  cells  and 
tissues  in  the  process.  Active  immunity  is  generally  of 
long  duration — some  months  at  least — and  is  not  trans- 
missible to  the  fetus  ;  but  passive  immunity  is  of  short 
duration — two  to  four  weeks — and  is  transmissible  to  the 
fetus  and  nursling.  Acquired  immunity  to  toxins  may  be 
due  to  the  elimination  of  the  receptors  concerned  in  the 
fixation  of  the  toxin  by  the  cells,  or  to  the  production  of 
the  neutralising  antitoxin.  The  leucocytes  are  probably 
the  active  agents  in  destroying  and  eliminating  toxin, 
whether  neutralised  by  antitoxin  or  not. 

Various  explanations  have  been  given  of  the  production 
of  acquired  immunity  against  the  organisms.  Pasteur 
suggested  that  the  organism,  by  its  growth  in  the  body, 
exhausts  some  specific  pabulum  necessary  for  its  develop- 
ment, so  that  it  cannot  again  grow  in  the  animal  which 
has  been  attacked.  This  hypothesis,  therefore,  pre- 
supposes that  in  the  body  there  is  some  nutrient  material 
necessary  for  the  growth  of  each  species,  which  is  difficult 
to  believe,  and  is  negatived  by  the  fact  that  an  organism 
will  grow  in  the  blood  and  tissues  removed  from  an  animal 
vaccinated  against,  and  insusceptible  to,  the  disease 
produced  by  itself. 

Pasteur's  '"  exhaustion  "  theory  has  been  revived  by  Ehrlich  1 
in  a  modified  form,  under  the  name  of  "  atrepsy,"  to  explain  certain 
cases  of  immunity.  Thus,  for  a  chemical  poison  to  act,  Ehrlich 
assumes  that  particular  receptors  in  the  protoplasm  for  binding 
the  poison  are  necessary  ;  these  he  terms  "  chemo -receptors." 
Bird-pox,  virulent  for  both  fowl  and  pigeon,  if  passed  through  the 
pigeon  becomes  completely  avirulent  for  the  fowl.  To  explain  this 
Ehrlich  suggests  that  the  parasite  in  passing  through  the  pigeon 
has  to  assimilate  substances  different  from  those  assimilated  during 
its  passage  through  the  fowl  ;  therefore  that  part  of  the  receptors 
which  deals  with  the  nutritive  substances  of  the  fowl's  organism  is 

1  "  Harben  Lecture,"  ii,  Journ.  Roy.  Inst.  Public  Health,  1907. 


IMMUNITY  207 

not  in  use  during  the  passage  through  the  pigeon,  and  may  become 
atrophied,  so  that  on  the  parasite  being  transferred  back  to  the 
fowl  it  will  not  be  able  to  thrive  owing  to  the  loss  of  the  receptors 
necessary  to  assimilate  the  fowl's  nutritive  substances.  Ehrlich 
suggests  that  the  majority  of  non -pathogenic  micro-organisms,  if 
introduced  into  the  animal  body,  perish  by  this  mechanism.  In 
the  case  of  mouse  carcinoma  inoculated  into  rats,  the  tumour-cells 
proliferate  for  a  few  days,  then  atrophy  and  disappear.  Ehrlich 
suggests  that  some  specific  substance  is  necessary  for  the  prolifera- 
tion of  mouse  carcinoma-cells  which  is  not  present  in  the  rat,  and 
as  soon  as  the  traces  of  this  specific  substance  carried  over  by  the 
inoculation  are  used  up,  the  cancer-cells  cease  to  proliferate  and 
finally  atrophy  and  disappear.  These  are  examples  of  Ehrlich's 
"  atrepsy  "  and  "  atreptic  immunity." 

Chauveau,  in  his  retention  theory,  suggested  that  the 
bacteria  during  their  growth  in  the  tissues  form  substances 
which  ultimately  inhibit  their  growth,  and,  if  the  animal 
recovers,  prevent  a  subsequent  development  of  the  organ- 
ism. The  same  objections  may  be  urged  against  this 
hypothesis  as  against  Pasteur's  exhaustion  hypothesis. 

Bacteriolysis  and  phagocytosis  are  probably  the  two 
main  factors  which  bring  about  the  refractory  condition 
in  acquired  immunity  against  bacteria,  as  well  as  recovery 
from  an  infection.  After  immunisation  it  may  be  shown 
that  phagocytosis  is  increased,  and  that  positive  chemotaxis 
takes  place  towards  the  organism,  whereas  previously 
negative  chemotaxis  occurred ;  the  leucocytes  have  been 
"  educated,"  as  it  were,  to  be  attracted,  instead  of  repelled, 
by  the  bacterial  invasion.  According  to  Andrewes,1  the 
defence  against  the  pyogenic  cocci  is  not  only  essentially 
phagocytic,  and  dependent  upon  the  polynuclear  leuco- 
cytes, but  is  also,  in  the  main,  opsonic.  In  tuberculosis 
and  syphilis  the  polynuclear  leucocyte  takes  little  part  in 
bodily  defence,  which  is  essentially  a  function  of  the  endo- 
thelial  and  fixed  tissue- cells.  With  the  colon  group  of 
organisms  certain  humoral  responses,  notably  agglutination 

1  "  Croonian  Lectures,"  Lancet,  June  25  et  seq.,  1910. 


208  A  MANUAL  OF  BACTERIOLOGY 

and  bacteriolysis,  are  better  marked  than  with  most  other 
bacteria,  and  polynuclear  phagocytosis  seems  subsidiary. 

Antitoxin  formation  probably  plays  little  or  no  part 
in  acquired  immunity,  or  even  in  recovery  from  infection. 
In  diphtheria,  for  instance,  antitoxin  is  not  found  until 
the  disease  has  subsided.  Possibly,  in  chronic  infections, 
antitoxin  formation  does  play  a  subsidiary  role  in  recovery. 

To  sum  up,  natural  immunity  is  probably  due  to  a 
number  of  factors,  some  or  all  of  which  may  be  operative 
in  particular  instances,  and  it  is  impossible  to  state  with 
certainty  any  general  law.  In  most  cases  phagocytosis 
is  the  principal  means  of  defence,  the  germicidal,  inhibi- 
tory, or  bacteriolytic  actions  of  the  body-fluids  aiding, 
though  of  subsidiary  importance ;  in  others  the  cells  and 
tissues  are  unaffected  by  the  bacterial  toxins,  sometimes 
because  the  cells  are  lacking  in  the  particular  side- chains 
or  receptors  which  fix  the  toxin ;  sometimes  because,  for 
some  unknown  reason,  the  cells  are  unaffected  by  the 
toxophore  group  of  the  toxin. 

As  regards  the  immunity  acquired  after  an  attack  of 
disease,  this  may  be  due  to  the  "  education  "  of  the  leuco- 
cytes, whereby  they  are  attracted,  whereas  formerly 
repelled,  by  the  products  of  bacterial  development,  or  to 
substances  which  stimulate  the  action  of  the  leucocytes. 
The  germicidal,  inhibitory,  and  bacteriolytic  actions  of  the 
body-fluids  may  also  be  enhanced.  It  seems  probable 
also  in  certain  instances  that  the  side- chains  or  receptors 
having  an  affinity  for  the  toxin  become  in  some  way 
destroyed  or  used  up,  so  that  further  fixation  of  the 
particular  toxin  cannot  take  place. 

It  is  to  be  noted,  as  Metchnikofl:  has  pointed  out,  that 
immunity  is  much  more  rapidly  acquired  against  micro- 
organisms than  against  their  toxins.  In  Nature,  it  is 
principally  against  micro-organisms  that  the  body  requires 
protection. 


PHAGOCYTOSIS  209 

Adaptability  seems  to  be  one  of  the  innate  properties 
of  protoplasm,  and  immunity  is  but  an  instance  of  adapta- 
bility. It  might  be  expected,  therefore,  that  immunity 
towards  infection  will  become  established,  more  or  less 
completely,  when  the  need  for  it  arises  ;  and  we  find  that 
this  is  the  case,  however  difficult  it  may  be  to  explain  the 
mechanism  by  which  it  is  attained. 

The  Role  of  the  Serum  in  Phagocytosis 

The  fact  that  in  an  immunised  animal,  no  sooner  does 
the  virulent  organism  gain  access  than  the  leucocytes 
migrate  to  the  site  of  infection,  surround  the  invaders, 
ingest  and  so  destroy  them,  was  at  one  time  ascribed  by 
Metchnikoff  to  "  education,"  i.e.  modification,  of  the 
leucocytes  ;  but  since  the  serum  of  the  immunised  animal 
injected  into  a  non- immunised  one  causes  the  leucocytes  in 
the  latter  to  behave  in  the  same  manner  as  they  do  in  the 
immunised  animal,  the  effect  must  be  due  to  something 
in  the  plasma  or  serum,  and  Metchnikoff  ascribed  the 
action  to  substances,  "  stimulins,"  which  heighten  the 
activity  of  the  leucocytes.  Later  work  has  not  confirmed 
this  view,  and  no  certain  proof  of  the  existence  of  stimulins 
is  forthcoming,  although  Leishman  attributed  a  stimulin 
action  to  thermostable  substances  in  the  serum  in  typhoid 
and  Malta  fevers.  Subsequently  Metchnikoff  conceived 
the  serum  as  acting,  not  on  the  leucocytes,  but  on  the 
microbe,  causing  it  to  become  positively  chemotactic  and 
no  longer  to  repel,  but  to  attract  the  phagocytes.  Con- 
siderable support  was  given  to  this  view  by  the  work  of 
Wright  and  Douglas,  who,  by  a  modification  of  Leishman's 
ingenious  method  for  quantitatively  estimating  phago- 
cytosis, emphasised  the  importance  of  the  serum  in  the 
mechanism  of  phagocytosis. 

Neufeld  and  Eimpau  also  concluded  that  substances, 


210  A  MANUAL  OF  BACTERIOLOGY 

"  bacteriotropines,"  are  produced  in  the  course  of  immu- 
nisation which  promote  the  phagocytosis  of  bacteria. 

Leishman's  method  for  estimating  phagocytosis* — A  thin  suspen- 
sion of  some  micro-organism,  e.g.  M.  pyogenes,  is  mixed  with  an 
equal  volume  of  blood  from  the  finger  ;  a  droplet  of  this  mixture 
is  placed  on  a  clean  slide,  and  covered  with  a  cover-glass,  and  the 
preparation  is  at  once  placed  in  a  moist  chamber  in  the  incubator 
at  37°  C.  for  half  an  hour.  At  the  end  of  this  time  it  is  taken  out, 
the  cover-glass  slipped  off,  and  the  films  on  slide  and  cover-glass 
are  driea,  fixed,  stained,  and  examined  microscopically,  and  the 
number  of  microbes  ingested  by  the  polymorphonuclear  leucocytes 
is  counted. 

Wright  and  Douglas  2  found  that  washed  leucocytes 
without  serum  are  non-phagocytic,  but  become  so  on  the 
addition  of  normal  serum.  If,  however,  the  serum  be 
first  heated  to  60°-65°  C.  before  being  added  to  the  mixture 
of  leucocytes  and  microbes,  phagocytosis  does  not  take 
place ;  but  if  the  unheated  serum  is  mixed  with  the  bac- 
teria, the  mixture  kept  at  37°  C.  for  fifteen  minutes  and 
then  heated  to  60°  C.  for  fifteen  minutes,  phagocytosis  can 
still  take  place,  thus  demonstrating  that  the  serum  acts 
in  some  way  on  the  bacteria,  rendering  them  suitable  prey 
for  the  phagocytes.  This  thermolabile  serum  feast  pre- 
parer  is  called  by  Wright  and  Douglas  "  opsonin  "  (from 
a  Greek  word  meaning  "  to  cater  for  "). 

They  have  also  shown  that  during  the  process  of  active 
immunisation  the  opsonic  value  of  the  serum  is  increased, 
and  they  have  succeeded  in  demonstrating  this  opsonic 
immunity  for  a  number  of  infections,  such  as  the  staphy- 
lococcic,  Malta  fever,  pneumococcic,  and  tuberculous.  If 
it  be  desired  to  measure  the  quantity  of  opsonins  present, 

1  Brit.  Med.  Jcurn.,  1902,  vol.  i,  p.  73. 

2  Prcc.  Roy.  See.  Lond.,  B.  Ixxii,  1903,  p.  357  ;  B.  Ixxiii,  1904,  p.  128  ; 
B.  Ixxiv,  1905,  pp.  147,  159  ;  B.  Ixxvii,  1907,  p.  211.  Also  in  Practitioner, 
May  1908  ;    various  papers  in  Lancet  and  Brit.  Med.  Journ. ;    Wright; 
fitudies  in  Immunity,  1909. 


OPSONINS  211 

say  in  a  case  of  furunculosis,  which  is  almost  always  caused 
by  the  M.  pyogenes,  the  following  are  required  :  (1)  a 
drop  or  so  of  the  patient's  serum  ;  (2)  a  drop  of  serum 
from  a  normal  person ;  (3)  a  suspension  in  salt  solution, 
of  a  culture  of  M.  pyogenes  preferably  derived  from  the 
furuncle ;  (4)  leucocytes  washed  free  from  the  plasma. 
Equal  volumes  of  the  patient's  serum,  leucocytes,  and 
suspension  are  mixed,  draw^n  up  in  a  capillary  tube,  incu- 
bated for  fifteen  minutes  at  37°  C.,  and  films  are  then 
prepared  and  stained.  As  a  control  a  similar  mixture  is 
prepared  and  treated  in  the  same  way,  but  using  the  normal 
serum  instead  of  that  of  the  patient.  The  films  are  then 
examined,  and  the  number  of  cocci  taken  up  by,  say, 
fifty  leucocytes  is  counted  in  the  two  specimens,  and  a 
ratio  obtained.  Taking  the  figure  for  the  normal  serum 
as  1,  that  for  the  patient's  serum  will  probably  be  0-5  or 
0-6,  and  this  is  termed  the  "  opsonic  index  "  (see  below, 
p.  219). 

In  subacute  and  chronic  local  infections  the  opsonic 
value  of  the  serum  is  usually  diminished,  occasionally 
increased.  In  acute  infections  the  index  will,  as  a  rule, 
below  ;  in  chronic  infections  which  are  not  strictly  localised, 
e.g.  tuberculosis,  the  index  will  sometimes  be  low,  some- 
times high.  A  low  index  generally  indicates  an  infection, 
or  a  low  power  of  resistance  to  the  particular  organism, 
or  that  a  chronic  but  quiescent  infection  exists ;  a  high 
index  may  indicate  that  the  person  has  had  an  infection 
but  has  overcome  it,  or  has  a  quiescent  infection.  The 
normal  index  for  healthy  persons  varies  only  within 
narrow  limits,  from  about  0-8  to  1-2  as  extremes  ;  an  index 
above  or  below  these  values  is  therefore  probably  patho- 
logical. 

By  injecting  small  quantities  of  a  vaccine  consisting  of 
a  killed  culture,  tuberculin,  etc.,  the  opsonic  index  can 
be  raised,  and  the  infection  thereby  tends  to  be  cured. 


212  A  MANUAL  OF  BACTERIOLOGY 

The  first  effect  of  the  injection  is  to  cause  a  fall  in  the 
opsonic  index,  the  "  negative  phase  "  of  Wright,  which 
is  usually  afterwards  followed  by  a  rise,  and  by  properly 
spacing  the  injections  a  considerable  rise  in  the  opsonic 
value  may  ultimately  result.  If  too  much  vaccine  be 
given  the  effect  may  be  to  permanently  depress  the  index 
and  cause  harm  instead  of  good,  hence  the  desirability  of 
controlling  all  injections  by  determinations  of  the  opsonic 
index.  This,  however,  renders  the  treatment  very  labo- 
rious, and  generally  by  employing  small  doses  and  allowing 
at  least  a  week  to  elapse  between  the  doses,  determina- 
tions of  the  opsonic  index  are  unnecessary  (for  dosage,  etc., 
see  p.  221).  By  movement,  massage,  etc.,  applied  at  or 
about  the  seat  of  a  local  infection,  bacterial  products  are 
disseminated  which  may  alter  the  index ;  a  process  of 
auto-inoculation  may  thus  result. 

The  opsonic  index  may  be  used  for  diagnostic  purposes  ; 
a  low  or  high  opsonic  value  towards  a  particular  organism 
suggests  that  an  infection  by  this  organism  exists  or  has 
recently  existed. 

Bulloch  came  to  the  conclusion  that  the  blood  contains 
a  number  of  specific  opsonins,  one  for  tubercle,  another  for 
M.  pyogenes,  and  so  on.  Simon,  Lamar,  and  Bispham,1 
however,  from  a  number  of  carefully  devised  experiments, 
conclude  that  specificity  of  opsonins  does  not  exist,  and 
suggest  that  opsonins  may  be  a  constant  quantity,  and 
that  the  number  of  organisms  taken  up  by  the  leuco- 
cytes is  influenced  by  a  second  unknown  and  variable 
factor. 

Russell 2  also  concludes  that  in  normal  serum  the  opsonins 
are  "  common  "  and  not  specific,  \nnd  can  be  removed  by 
a  number  of  bodies.  In  immune  serum,  on  the  other 
hand,  both  "common"  and  "immune"  opsonins  are 

1  Journ.  Exper.  Med.,  vol.  viii,  1906,  p.  651. 

2  Johns  Hopkins  Hosp.  Bull.,  vol.  xviii,  1907,  p.  252. 


THE  OPSONIC  METHOD  213 

present,  the  latter  being  quite  specific.  That  is  to  say,  in 
the  process  of  immunisation  specific  opsonins  are  formed 
and  the  increase  of  opsonins  following  injection  of  a  vaccine 
is  probably  due  to  the  formation  of  immune  opsonins  which 
react  specifically. 

Muir  and  Martin 1  believe  that  in  immune  serum  a  specific, 
immune,  thermostable  opsonin  is  present,  and  also  a 
normal,  thermolabile  opsonin. 

Wright  considers  the  opsonins  to  be  substances  distinct 
from  all  others,  but  MetchnikofT,  Dean,  and  other  observers 
suggest  that  they  are  identical  with  the  "  substance 
sensibilisatrice." 

It  is  doubtful  if  opsonins  are  present  in  more  than 
traces  in  the  unaltered  blood  plasma  :  like  alexins,  they 
seem  to  develop  as  a  result  of  coagulation.  The  role  of 
opsonins  in  immunity  and  in  recovery  from  infection  is 
therefore  a  complex  problem. 

The  opsonic  method  has  been  criticised  of  late.  Thus  Moss2 
says  :  "  None  of  the  present  methods  of  estimating  the  opsonic 
content  of  the  blood  seems  sufficiently  accurate  to  be  of  practical 
value  "  ;  Fitzgerald,  Whiteman,  and  Strangeways,3  in  an  elaborate 
investigation,  concluded  that  the  method  is  unreliable.  Whereas 
Wright  takes  into  account  the  serum  only,  Shattock  and  Dudgeon  4 
state  that  "  the  cells  (i.e.  the  phagocytes)  vary  in  value  like  the 
serum."  It  may  be  granted  that  the  whole  truth  respecting  the 
opsonic  reaction  and  method  is  not  yet  fully  known,  but  many  of 
the  criticisms  have  been  based  on  an  imperfect  technique.  On  the 
whole,  it  may  be  said  that  Wright's  method,  with  careful  technique 
and  in  practised  hands,  gives  information  previously  impossible  to 
obtain,  and  the  proper  dosage  of,  and  treatment  by,  vaccines  has 
been  largely  elaborated  by  means  of  it. 

1  Proc.  Roy.  Soc.  Lond.,  B.  Ixxix,  1903,  p.  187. 

2  Johns  Hopkins  Hosp.  Bull.,  vol.  xviii,  1907,  p.  237. 

3  Bull.  Committee,  for  the  Study  of  Special  Diseases  (Cambridge),  vol.  i, 
1907,  No.  8. 

4  Proc.  Roy.  Soc.  Med.,  vol.  i,  1908,  "  Medical  Section,"  p.  169. 


214  A  MANUAL  OF  BACTERIOLOGY 

Method  of  Determining  the  Opsonic  Index 

The  requisites  are  : 

1.  Several  Wright's  pipettes  with  india-rubber  teats. 

2.  The  serum  of  the  patient  to  be  tested. 

3.  The  serum  of  a  healthy  person  for  a  control. 

4.  A  suspension  of  the  organism  for  which  the  deter- 
mination is  to  be  made. 

5.  A  suspension  of  living  leucocytes. 

1.  Wright's  pipettes  with  india-rubber  teats. — These  are 
made  of  glass  tubing  of  the  form  shown  in  a,  Fig.  35, 
which  is  about  two- thirds  full  size.  Glass  tubing  must  be 
chosen  which  properly  fits  the  teats.  A  piece  of  glass- 
tubing  about  4  inches  in  length  is  taken,  heated  in  the 
blowpipe  flame  until  quite  soft,  then  it  is  taken  out  of 
the  flame  and  the  two  ends  are  drawn  steadily  apart ; 
the  more  they  are  drawn  apart,  the  finer  will  be  the  bore 
of  the  tube — about  ^  in.  is  a  suitable  size.  The  middle 
of  the  capillary  part  should  then  be  introduced  into  a 
small  white  gas- flame  and  drawn  apart  so  as  to  form 
two  pipettes.  By  filing  off  the  sealed  end  at  a  suitable 
spot  the  open  extremity  may  be  slightly  contracted  as 
shown  in  b  ;  this  prevents  the  column  of  fluid  in  the  tube 
moving  so  quickly. 

2  and  3.  The  sera. — These  two  specimens  should  be 
taken  at  about  the  same  time,  and  the  determination 
should  be  made  as  soon  as  possible. 

The  blood  is  preferably  collected  in  a  Wright's  capsule 
(Fig.  35,  d).  Both  ends  of  the  pipette  are  broken  off, 
and  the  blood  is  collected  by  immersing  the  bent  end  in 
the  blcod  as  it  luns  from  a  prick  with  a  Hagedorn  or 
triangular  needle  in  the  ear  or  finger.  The  capsule,  which 
should  be  at  least  one-third  filled,  is  then  sealed  in  the 
flame,  the  dry  or  straight  end  being  sealed  first.  After 


THE  OPSONIC  INDEX 


215 


coagulation,  which  may  be  hastened  by  placing  in  the 
warm  incubator  for  half  an  hour,  the  capsule  is  hung  Ly 
the  curved  end  in  the  centrifuge  and  centrifuged  to  obtain 
clear  serum.  Little  change  in  the  serum  ensues  for  two 
to  three  days  if  the  capsules  are  kept  sealed.  The  capsules 
may  be  stuck  into  a  lump  of  plasticine  until  required. 


FIG.  35. — a.  Glass  pipette,  with  india-rubber  teat  for  opsonic 
determinations,  etc.  ;  6  shows  (enlarged)  the  contracted 
extremity  of  the  pipette  ;  c  shows  the  stem  of  the  pipette, 
containing  the  equal  volumes  of  serum,  leucocytic  suspension, 
and  bacterial  suspension,  before  mixing ;  d  is  the  Wright's 
capsule  for  collecting  blood. 

Plasticine  is  useful  for  many  such  purposes,  for  temporarily 
plugging  tubes,  etc. 

4.  Suspension  of  the  organism. — In  the  case  of  tubercle, 
suitable  dead  cultures  can  be  purchased.  To  prepare 
the  suspension  from  these,  a  small  portion  of  the  growth 
(about  as  big  as  a  grain  of  rice)  is  ground  up  in  a  small 
agate  mortar,  1-5  per  cent,  salt  solution  being  added  drop 
by  drop  up  to  2  c.c.  This  suspension  will  still  contain 
clumps,  which  must  be  got  rid  of  by  centrifuging  for  three 
or  four  minutes.  With  the  tubercle  bacillus  and  gono- 
coccus  spontaneous  phagocytosis  is  apt  to  occur  if  ordinary 
(0-8  per  cent.)  salt  solution  is  used. 

A  staphylococcic  suspension  is  prepared  by  taking  an 
agar  culture  not  more  than  twenty- four  hours  old,  adding 
salt  solution  (0-8  per  cent.),  and  shaking  gently  so  as  to 
wash  off  the  growth.  When  the  suspension  is  made  it 


216  A  MANUAL  OF  BACTERIOLOGY 

must  be  pipetted  off  into  a  small  tube  and  centrifuged  for 
a  few  minutes.  The  suspension  must  not  be  too  thick, 
otherwise  the  leucocytes  will  take  up  an  unaccountable 
number  of  cocci ;  the  proper  density  can  be  judged  by 
experience  alone,  but  the  suspension  should  be  only  faintly 
opalescent.  Suspensions  of  pneumococci  and  other  organ- 
isms are  made  in  the  same  way.  Variations  in  the  number 
of  bacteria  ingested  may  occur  according  as  recently  isolated 
or  old  strains  are  employed. 

Instead  of  centrifuging,  the  suspensions  may  be  filtered 
through  a  double  thickness  of  filter-paper. 

5.  Suspension  of  living  leucocytes. — To  prepare  this, 
take  about  10  c.c.  of  physiological  salt  solution  containing 
J  per  cent,  of  sodium  citrate,  to  prevent  the  coagulation 
of  the  blood.  This  must  be  freshly  prepared  (or  kept 
sterile,  which  is  inconvenient),  and  the  simplest  method 
is  to  use  "  soloids  "  prepared  for  the  purpose  by  Burroughs 
and  Wellcome  ;  one  of  these  dissolved  in  10  c.c.  of  distilled 
water  will  yield  the  solution  required.  This  is  put  into 
a  centrifuge  tube  and  warmed  to  blood-heat.  A  healthy 
person  is  then  pricked  in  the  ear  or  finger,  and  his  blood 
is  allowed  to  drop  into  the  fluid  until  1  c.c.  or  more  has 
been  collected.  The  tube  is  then  centrifuged  until  all  the 
corpuscles  have  come  to  the  bottom  and  the  supernatant 
fluid  is  left  clear.  If  the  deposit  is  closely  examined  the 
red  corpuscles  will  be  seen  to  be  at  the  bottom,  whilst 
above  them  there  is  a  thin  grey  layer  of  leucocytes.  The 
whole  of  the  clear  fluid  is  then  pipetted  off,  as  close  as 
possible  to  the  leucocyte  layer,  but  without  disturbing 
the  latter,  with  a  pipette  armed  with  an  india-rubber  teat, 
or  with  a  syringe.  The  tube  is  again  filled  with  saline 
solution,  the  blood  and  fluid  are  mixed,  the  mixture  is 
centrifuged,  and  the  clear  fluid  pipetted  off,  and  this 
process  of  washing  is  repeated.  Next,  the  leucocyte  layer 
with  the  upper  layer  of  red  corpuscles  (which  also  contains 


THE  OPSONIC  INDEX  217 

leucocytes)  is  pipetted  off  into  a  small  tube,  and  the  whole 
is  thoroughly  mixed  by  repeatedly  sucking  into,  and 
expelling  from,  the  pipette.  The  result  is  a  suspension  of 
living  leucocytes  mixed  with  red  corpuscles. 

The  process.- — (1)  Make  a  pipette  and  place  an  india- 
rubber  teat  on  the  thick  end.  With  a  grease  pencil  or 
with  ink,  make  a  transverse  line  about  an  inch  from  the 
point ;  the  volume  of  fluid  contained  between  the  point 
and  this  mark  is  spoken  of  as  the  unit. 

(2)  Having  the  patient's  serum  and  the  suspensions  of 
leucocytes  and  of  bacteria  ready  to  hand,  take  the  pipette 
between  the  index  ringer  and  thumb  of  the  right  hand 
and  compress  the   nipple.     Immerse  the  point  beneath 
the  surface  of  the  suspension  of  bacilli,  and  relax  the 
pressure  on  the  nipple  until  the  suspension  has  risen  exactly 
to  the  mark  so  that  one  unit  has  been  drawn  up  ;    then 
remove  the  point  from  the  fluid  and  relax  the  pressure 
again  so  that  a  small  volume  of  air  is  sucked  up.     This 
will  be   quite  easy  if  the  point  is  a  good  one,  otherwise 
it  will  be  difficult  or  impossible,  as  the  column  of  fluid 
will  either  refuse  to  stir  or  will  oscillate  violently.     Next 
immerse  the  point  in  the  suspension  of  leucocytes  and  draw 
up  one  unit.     This  will  be  separated  from  the  suspension 
of  bacteria  by  the  bubble  of  air.     Kemove  the  point  from 
the  suspension  and  draw  up  a  second  volume  of  air. 

Lastly,  draw  up  one  unit  of  the  serum.  There  will  now 
be  in  the  pipette  (counting  from  the  nipple  towards  the 
point)  one  unit  of  bacterial  suspension,  a  bubble  of  air,  a 
unit  of  leucocytes,  a  bubble  of  air,  and  lastly  a  unit  of 
serum  (c,  Fig.  35). 

(3)  Put  the  point  of  the  pipette  on  to  a  clean  hollow- 
ground  slide  or  an  artist's  porcelain  sunk  palette,   and 
express  the  whole  of  its  contents,  and  mix  well  together, 
aspirating  them  repeatedly  into  the  pipette  and  expelling 
without  causing  bubbles.    If  bubbles  form,  a  hot  wire 


218  A  MANUAL  OF  BACTERIOLOGY 

brought  near  will  quickly  dispel  them.  When  thoroughly 
mixed,  aspirate  the  mixture  into  the  pipette,  suck  up  a 
short  volume  of  air,  and  seal  the  tip  in  the  flame. 

Then  place  the  pipette  point  downwards  in  the  incubator, 
or  better,  in  a  water-bath  at  35°  to  37°  C.,  noting  the 
time  exactly,  and  proceed  to  prepare  a  second  pipette  in 
precisely  the  same  way,  using  the  same  suspensions  of 
bacteria  and  leucocytes,  but  the  control  serum  instead  of 
the  patient's.  Place  this  in  the  incubator  or  water-bath, 
by  the  side  of  the  other,  noting  the  time  at  which  this  is 
done.  When  each  pipette  has  been  incubated  for  a  quarter 
of  an  hour  it  is  removed  from  the  incubator  or  water-bath, 
the  end  broken  off  and  the  nipple  fitted  to  the  thick  end  ; 
then  the  contents  are  expelled  on  to  a  hollow  slide  or 
porcelain  palette  and  mixed  thoroughly  together.  Films 
are  then  prepared.  This  may  be  done  by  depositing  a 
drop  in  the  middle  of  a  large  cover- glass  (1-inch  squares, 
No.  2),  dropping  on  to  it  another  cover- glass  and  drawing 
the  two  apart.  Or  the  films  may  be  made  on  slides,  for 
which  Wright  recommends  roughing  the  slides  with  fine 
emery  paper  and  spreading  the  film  with  the  sharp  edge 
of  a  broken  slide  (see  next  page).  The  films  then  have  to 
be  stained.  For  staphylococci,  streptococci,  pneumococci, 
B.  coli,  etc.,  the  films  may  be  fixed  with  formalin  and 
stained  with  carbol-thionine  blue  or  borax-methylene  blue 
(see  "  Malaria  "),  or  they  may  be  stained  without  previous 
fixing  with  the  Leishman  stain.  For  tubercle,  the  films 
may  be  fixed  in  a  saturated  solution  of  mercuric  chloride 
(one  or  two  minutes),  stained  in  warm  carbol  fuchsin, 
decolorised  with  2J  per  cent,  sulphuric  acid  in  methylated 
spirit,  and  counterstained  with  methylene  blue. 

Wright  now  uses  the  whole  blood  instead  of  the  leuco- 
cyte layer  only.  After  the  blood  has  been  drawn  into  the 
titrated  salt  solution  it  is  centrifuged,  washed  twice  with 
salt  solution,  the  fluid  is  pipetted  off,  and  finally  the 


PREPARATION  OF  VACCINES  219 

corpuscles  are  well  mixed.  The  various  mixtures — washed 
corpuscles,  bacterial  suspension,  and  serum — are  made 
and  incubated  as  previously  described.  In  order  to  make 
the  film  for  staining  and  counting,  the  contents  of  the 
pipette  are  discharged  on  to  one  end  of  a  slide  roughed  with 
fine  emery  paper  and  the  mixture  is  spread  by  means  of  a 
slide  which  has  been  broken  across  after  notching  with 
a  file  or  glass  cutter.  The  object  is  to  obtain  a  broken  edge 
having  a  very  slight  concavity,  and  many  slides  may 
have  to  be  sacrificed  to  attain  this.  The  film  is  spread  by 
drawing  (not  pushing)  along,  the  leucocytes  adhere  to 
the  edge  of  the  spreader,  and  finally  are  deposited  mostly 
at  the  end  of  the  preparation,  the  red  corpuscles  being 
left  behind. 

Lastly,  the  films  after  staining  are  examined  with  the 
oil-immersion  lens,  preferably  with  the  aid  of  a  mechanical 
stage,  and  the  number  of  organisms  contained  in  not  less 
than  fifty  polymorphonuclear  leucocytes  is  counted.  Parts 
of  the  film  in  which  the  cells  are  broken  down  or  not  well 
stained,  or  cells  containing  obvious  clumps  of  organisms, 
should  be  avoided.  The  ratio  between  the  number  in 
the  control  and  the  number  in  the  specimen  prepared  with 
the  patient's  serum  gives  the  opsonic  index.  Thus,  if  in 
the  control  there  are  125,  while  in  the  patient's  specimen 
there  are  75,  the  index  would  be  T7^5-  =  0-6,  i.e.  not  much 
more  than  half  the  normal. 

Preparation  of  vaccines  for  treatment,  etc. — The  vaccine  used  for 
treatment  is  a  sterilised,  standardised  suspension  of  the  infecting 
organism,  except  in  the  case  of  tuberculosis,  for  which  tuberculin 
(TR  or  BE)  or  an  analogous  preparation  is  employed.  In  certain 
instances  a  mixture  of  organisms  is  used — e.g.  M.  pyogenes,  var. 
aureus  and  var.  albus,  with  or  without  the  acne  bacillus  in  some 
cases  of  acne — and  the  strain  of  organism  isolated  from  the  lesion 
is  generally  to  be  preferred. 

The  vaccine  is  prepared  by  growing  the  organism  under  appro- 
priate conditions, ,  the  staphylococcus  on  agar,  the  streptococcus, 


220  A  MANUAL  OF  BACTERIOLOGY 

pneumococcus,  and  gonococcus  on  blood-agar,  etc.  The  growth  is 
then  made  into  a  suspension  by  adding  a  few  drops  of  sterile  0-1  per 
cent,  sodium  chloride  solution  and  well  rubbing  up  with  a  sterile  glass 
or  aluminium  rod.  Two  or  three  tubes  are  treated  in  this  way ; 
the  suspension  is  poured  into  a  sterile  tube  or  small  flask  of  stout 
glass,  the  culture  tubes  are  rinsed  out  with  a  little  more  of  the  salt 
solution,  and  the  washings  added  to  the  suspension,  two  or  three 
sterile  glass  beads  are  added,  and  the  vessel,  sealed  or  corked,  is 
shaken  vigorously  for  some  time,  preferably  in  a  shaking  machine, 
so  as  thoroughly  to  break  up  the  masses  of  organisms.  The  con- 
tents of  the  vessel,  which  should  measure  5  c.c.  or  thereabouts,  are 
then  centrifuged  for  some  minutes,  the  suspension  is  poured  off  from 
the  deposit  into  a  second  sterile  flask  and  is  now  ready  for 
standardisation. 

Standardisation  is  carried  out  by  Wright's  method.  Two  or 
three  volumes  of  citrate  solution  are  sucked  up  into  a  pipette  such 
as  that  used  for  opsonic  determinations,  the  finger  is  pricked  and 
one  volume  of  blood  is  taken  up  in  the  pipette,  separated  from  the 
citrate  solution  by  an  air-bubble,  and  finally  one  volume  of  the 
bacterial  suspension,  also  separated  from  the  blood  by  an  air-bubble, 
is  taken  up.  The  whole  contents  of  the  pipette  are  then  well 
mixed  by  expelling  on  to  a  clean  slide  and  sucking  up  three  or  four 
times.  About  one-third  of  the  mixture  is  then  transferred  to  each 
of  three  clean  slides,  and  the  drops  are  spread  with  the  edge  of  a 
slide  so  as  to  obtain  thin  uniform  smears.  These  are  allowed  to 
dry,  stained  with  Leishman's  stain,  and  the  number  of  red  corpuscles 
and  bacteria  is  counted  in  a  number  of  microscopical  fields.  Assum- 
ing that  there  are  5,000,000  red  cells  in  a  cubic  millimetre  of  blood,  it 
is  easy  to  calculate  approximately  the  number  of  bacteria  contained 
in  the  suspension.  Suppose  that  500  red  cells  have  been  counted, 
and  with  these  1500  bacteria  are  admixed.  Since  equal  volumes 
of  blood  and  suspension  have  been  taken,  one  cubic  millimetre  of 

bacterial   suspension   will   contain  5'000>0^Q  X  15Q°  =  15,000,000 

£>00 

bacteria.  But  one  cubic  centimetre  contains  1000  cubic  milli- 
metres, therefore  the  suspension  contains  15,000,000  x  1000  = 
15,000,000,000  bacteria  per  cubic  centimetre,  and  by  appropriate 
dilution  any  bacterial  content  of  the  suspension  may  be  obtained. 
Thus,  if  1,000,000,000  organisms  per  cubic  centimetre  is  desired, 
1  c.c.  of  the  suspension  must  be  diluted  with  14  c.c.  of  salt  solution. 
To  the  prepared  dilution  of  the  bacterial  suspension  0-5  percent,  of 
carbolic  acid,  or  0-2  per  cent,  of  trikresol,  is  added,  and  the  flask  is 
placed  in  a  water-bath  at  56°  to  60°  C.  for  one  or  one  and  a  half 


DOSAGE  OF  VACCINES 


221 


hours,  according  to  the  resistance  of  the  organism.  The  stock 
solution  may  subsequently  be  introduced  into  small  sterile  glass 
ampoules  of  1-2  c.c.  capacity,  which,  after  sealing  and  standing  for 
twenty -four  hours,  may  again  be  sterilised  for  an  hour  at  60°  C. 
to  ensure  the  destruction  of  the  organisms  ;  cultures  may  be  made 
from  the  sterilised  vaccine  to  ascertain  that  this  is  the  case.  The 
lower  the  temperature  and  the  less  the  heating,  consistent  with 
sterilisation,  the  more  active  will  be  the  vaccine. 

The  annexed  Table  x  gives  an  idea  of  the  doses  of  vaccines,  their 
toxicity,  and  frequency  of  inoculation. 


Vaccine 

Relative  toxicity 

Doses 

Frequency  of 
inoculation 

Tuberculin 

Very  toxic 

looooo  ~~  foooo  ~ 

Every  10-14  days. 

B.  coli 

Very  toxic 

5-15  millions 

Every  2,  5,  or  10 

days. 

Pneumococcic 

Less  toxic 

10-50  millions 

Every  36-48 

than  B.  coli 

hours  in  pneu- 

monia ;    every  10 

days  in  chronic 

infections. 

Streptococcic 

More  toxic  than 

20-60  millions 

Every  7-14  days. 

pneumococcic 

Staphylo- 

Less  toxic  than 

100-1000 

Every  10  days. 

coccic 

streptococcic 

millions 

M.  melitensis 

— 

Y^Q-  sq.  cm.  of 

Every  7-14  days. 

surface  agar 

culture  (because 

very  difficult  to 

count) 

Gonococcic 

Slightly  toxic 

100-500  millions 

Every  7-14  days. 

The  smaller  doses  are  given  at  the  commencement  of  the  treat- 
ment, and  the  doses  are  gradually  increased. 

The  writer  has  employed  endotoxin  solutions  as  vaccines  and 
believes  they  are  very  efficient. 

Prophylactic  vaccines. — In  addition  to  the  therapeutic  vaccines 
for  the  treatment  of  the  declared  disease,  vaccines  are  also  employed 
for  prevention  of  disease.  The  preventive  or  prophylactic  vaccines 
may  be  : 

(1)  Living,  but  attenuated,  cultures,  e.g.  anthrax  and  cholera. 

1  See  Harris,  Practitioner,  May  1908,  p.  647. 


222  A  MANUAL  OF  BACTERIOLOGY 

This  method  has  also  been  proposed  for  plague,  and  vaccinia  must 
be  regarded  as  being  of  this  nature  (this  is  the  "  Pasteurian 
method  "). 

(2)  Killed  cultures,   autolysed  cultures,   and  endo-toxins. — The 
first  and  second  are  used  for  typhoid,  plague  and  dysentery,  and 
Hewlett  has  suggested  endo-toxins  for  typhoid,  cholera,  plague  and 
diphtheria. 

(3)  Immune  sera  give  protection  for  a  limited  time. 

(4)  Besredka   has   suggested    "  sensitised   vaccines,"    i.e.   living 
cultures  saturated  with  the  homologous  immune  body  derived  from 
an  immune  serum.     He  claims  that  the  organisms  being  unaltered 
by  heating,  etc.,  the  vaccine  gives  better  results  than  a  dead  vaccine, 
while  the  saturation  with  the  immune  serum  prevents  infection 
although  the  organisms  are  living. 

(For  further  particulars,  see  Hewlett's  Serum  Therapy,  ed.  2, 
J.  and  A.  Churchill,  1910.) 


CHAPTER  VI 
SUPPURATION  AND  SEPTIC  CONDITIONS 

THE  subjects  of  septic  infection  and  of  suppuration  are  of 
great  practical  importance,  and  a  knowledge  of  their 
etiology  is  one  of  the  main  factors  which  have  conduced 
to  the  great  advances  that  were  made  during  the  Victorian 
era  in  the  treatment  of  wounds,  whether  accidental  or 
made  by  the  surgeon's  knife. 

Ogston  in  1881  and  Rosenbach  in  1884  demonstrated 
that  micro-organisms  are  almost  invariably  present  in 
the  pus  of  acute  abscesses,  and  these  observations  were 
repeatedly  confirmed  by  subsequent  investigators.  A 
number  of  experiments  were  then  initiated  in  order  to 
ascertain  whether  these  organisms  bear  a  causal  relation 
to  the  phenomena  of  suppuration  or  are  merely  accidenta  ly 
present.  These  experiments  showed  that  a  large  number 
of  organisms  can  produce  suppuration,  and  render  it 
certain  that  in  ninety-nine  cases  out  of  a  hundred  the 
suppurative  and  septic  conditions  met  with  spontaneously, 
or  occurring  after  surgical  interference,  are  due  to  the  action 
of  micro-organisms.  The  chief  of  these  are  several  micrococci 
(commonly  known  as  staphylococci,  and  the  infections  which 
they  produce,  as  staphylococcic  infections)  and  streptococci. 

Under  the  terms  "  suppuration  "  and  "  septic  diseases  " 
are  included  such  varied  conditions  as  abscesses,  boils  and 
carbuncles,  cellulitis,  osteomyelitis,  erysipelas,  gonorrhoea, 
infective  endocarditis,  pyaemia,  septica3mia  and  saprsemia, 
puerperal  fever,  and  hospital  gangrene. 

223 


224  A  MANUAL  OF  BACTERIOLOGY 

As  will  be  gathered  from  the  descriptions  of  the  individual 
organisms,  suppuration  may  be  set  up  by  inoculation  with 
several  species,  and  a  number  of  experiments  by  various 
observers,  carried  out  by  inunction,  subcutaneous  inocu- 
lation, and  inoculation  in  the  serous  cavities  and  circula 
tion,  have  conclusively  proved  that  this  is  the  case,  not 
only  in  animals,  but  also  in  man. 

A  problem  of  great  importance  is  whether  micro- 
organisms are  usually  the  cause  of  suppuration,  or  whether 
mechanical  injury,  chemical  agents,  etc.,  can  also  produce 
it.  Mechanical  injury  alone  does  not  seem  to  be  capable 
of  inducing  pus  production,  but  it  is  otherwise  with  regard 
to  chemical  agents.  For  a  long  time  considerable  differ- 
ence of  opinion  existed  and  discordant  results  were 
published.  These  discrepancies  have  now  been  explained, 
and  are  found  to  depend  upon  the  method  of  experiment 
and  the  particular  animal  and  chemical  agent  employed 
That  chemical  agents  should  produce  suppuration  might 
be  expected,  for  it  would  be  against  analogy,  derived  from 
all  other  bacterial  diseases,  if  the  pyogenic  organisms  do 
not  produce  suppuration  through  the  chemical  substances 
formed  by,  or  present  within,  their  cells,  and  if  these 
chemical  substances  act  thus,  why  should  not  other 
chemical  substances  be  found  to  act  in  a  similar  way  ? 

In  experiments  with  chemical  agents  the  greatest  care 
has  to  be  taken  to  exclude  the  entrance  of  micro-organisms. 
This  is  best  done  by  sealing  the  sterilised  substance  in 
sterilised  fusiform  glass  tubes  and  introducing  these  under 
the  skin  or  into  the  tissues  with  strict  aseptic  precautions. 
When  the  wounds  have  completely  healed  the  tubes  are 
broken  by  pressure  and  their  contents  allowed  to  diffuse 
nto  the  surrounding  tissues. 

Sterilised  cultures  (above  a  certain  amount)  of  the 
Micrococcus  pyogenes  and  a  crystalline  body,  phlogosin, 
obtained  by  Leber  from  its  cultures,  produce  abscesses  on 


SUPPURATION  225 

inoculation.  Mercury  produces  suppuration  in  the  dog, 
but  not  in  the  rabbit ;  silver  nitrate  (5  per  cent,  solution) 
has  a  similar  action.  Ammonia  fails  to  produce  pus ;  it 
is  either  absorbed  without  damage,  or  if  in  stronger  solution 
produces  necrosis  of  the  tissues.  Turpentine  produces 
large  sterile  abscesses  in  carnivora,  and  Brieger's  cadaverine 
is  likewise  stated  to  set  up  suppuration. 

Buchner  was  also  able,  by  warming  various  bacteria 
with  0-5  per  cent,  caustic  potash,  to  obtain  a  solution 
containing  protein  which  was  powerfully  pyogenic,  and 
Nannotti  found  that  sterilised  pus  had  a  similar  property. 
It  thus  seems  certain  that  a  number  of  chemical  sub- 
stances can  set  up  suppuration.  At  the  same  time,  it 
must  be  clearly  recognised  that  suppuration  and  sup- 
purative  complications,  as  they  occur  naturally,  are  to  be 
regarded  as  due  to  the  activity  of  micro-organisms  in 
almost  every  instance. 

Of  so-called  "  septic  "  diseases,  sapraemia,  septicaemia, 
and  pyaemia  must  be  mentioned.  By  "  sapraemia "  is 
meant  the  constitutional  condition  arising  from  the 
absorption  of  the  toxic  products  elaborated  by  micro- 
organisms, the  latter  being  localised  and  absent  from 
the  general  circulation.  In  the  acute  form  it  is  not  a 
common  condition,  the  best  example  being  that  which 
occurs  after  parturition ;  by  simply  clearing  and  washing 
out  the  uterus  the  symptoms  rapidly  abate.  In  septicaemia 
not  only  is  there  usually  (though  not  necessarily)  a  local 
site  of  infection,  but  in  addition  micro-organisms  are 
present  in  the  general  circulation.  It  is  true  they  are  not 
abundant  in  the  latter  situation,  and  Cheyne1  believes 
that  they  are  to  a  large  extent  arrested  in  the  capillaries. 
Micrococci  and  streptococci  are  the  commonest  forms. 
Pyaemia  is  characterised  by  the  presence  of  micro-organisms, 
most  frequently  streptococci,  in  the  general  circulation, 

1  System  of  Medicine,  Clifford  Allbutt,  ed.  2,  vol.  i,  p.  876. 

15 


226  A  MANUAL  OF  BACTERIOLOGY 

and  in  addition  by  the  formation  of  abscesses  in  various 
situations.  These  arise  usually  from  suppurative  phlebitis 
with  the  formation  of  septic  emboli  and  thrombi.  The 
sequence  of  events,  according  to  Cheyne,1  is  (a)  phlebitis 
in  direct  connection  with  the  wound ;  (b)  a  thrombus 
impregnated  with  micro-organisms  is  formed  in  the  vein  ; 
(c)  this  softens  and  disintegrates,  and  particles  or  emboli 
are  carried  to  distant  parts ;  (d)  these  lodge  in  the 
capillaries,  with  the  formation  of  infarctions  and  abscesses. 
Suppurative  pylephlebitis  is  a  pyaemia  affecting  the  portal 
system  of  vessels.  As  regards  the  so-called  chronic 
pyaemia  or  multiple  abscesses,  Cheyne  considers  that  it 
differs  from  true  pyaemia  in  that  embolism  plays  no  part. 
Organisms  gain  access  to  the  blood- stream,  settle  in  any 
spot  where  the  vitality  of  the  tissues  is  depressed,  grow 
and  multiply,  and  there  produce  an  abscess. 

The  mere  presence  of  micro-organisms  does  not  always 
suffice,  however,  for  they  may  be  present  without  pro- 
ducing suppuration  ;  and  the  same  organism,  for  example, 
the  Streptococcus  pyogems,  may  at  one  time  produce  a 
localised  abscess,  at  another  diffuse  cellulitis.  and  at  a 
third  pyaemia  ;  a  number  of  factors  control  and  modify 
the  occurrence  and  the  particular  form  of  septic  disease. 

As  already  mentioned  (p.  199),  many  micro-organisms 
when  injected  into  the  blood-stream  are  rapidly  disposed 
of ;  so  when  moderate  quantities  of  the  Micrococcus 
pyogenes  are  injected  into  the  circulation  of  a  rabbit, 
abscesses,  as  a  rule,  form  only  in  the  kidney.  If,  however, 
the  organisms  be  attached  to  gross  particles,  so  that  they 
cannot  pass  through  the  capillaries,  embolism  occurs  and 
abscesses  form  about  the  embolic  foci.  The  virulence  of 
.the  infecting  organism,  which  varies  much,  is  another  factor 
of  great  importance.  The  effect  of  inflammation  and 
injury  in  making  a  part  "  susceptible  "  is  also  very  marked. 
1  Loc.  cit.  p.  881. 


MICROCOCCUS  PYOGENES  227 

Inject  the  M.  pyogenes  into  animals  in  which  the  endo- 
cardium or  a  bone  has  been  damaged,  and  in  all  probability 
an  endocarditis  or  an  osteomyelitis  will  ensue.  The  dose 
and  concentration  of  the  organisms  are  also  important 
factors.  Watson  Cheyne  found  that  250,000,000  cocci 
(M.  pyogenes)  injected  into  the  muscles  of  a  rabbit  pro- 
duced a  circumscribed  abscess,  but  1,000,000,000  caused 
a  general  septicaemia  and  death.  So,  probably,  while  the 
cells  in  a  healthy  wound  can  dispose  of  a  few  organisms, 
if  the  latter  are  abundant  or  in  masses  they  may  gain  the 
mastery. 

Micrococcus   pyogenes,    var.    aureus   (Staphylo- 
coccus  pyogenes  aureus) 

Morphology  and  biology. — A  minute  spherical  organism 
measuring  about  0'75  //.  in  diameter.  It  generally  occurs 
in  more  or  less  irregular  groups,  but  may  be  met  with 
singly  or  in  pairs  (Plate  I.  c).  It  is  non-motile,  does  not 
form  spores,  and  stains  well  with  all  the  anilin  dyes  and 
also  by  Gram's  method.  It  is  aerobic  and  facultatively 
anaerobic,  will  develop  in  vacuo,  and  grows  well  and 
rapidly  on  all  the  usual  culture  media  at  temperatures 
from  18°  to  37°  C.  On  agar-agar  it  forms  a  thickish, 
moist,  shining  growth,  cream-coloured  at  first,  but  after 
a  day  or  two  developing  a  characteristic  orange-yellow 
colour.  It  grows  in  the  same  manner  on  blood-serum 
without  liquefaction  of  the  medium.  Gelatin  is  rapidly 
liquefied,  the  liquefied  gelatin  being  at  first  somewhat 
turbid  from  yellowish  masses  of  organisms  ;  these  later 
on  subside  and  form  an  orange-yellow  sediment  (Plate 
I.  d)  In  gelatin  plates  the  colonies  form  at  first  small 
whitish,  granular  points,  developing  in  two  or  three  days 
into  circular  areas  of  liquefaction  with  yellowish  masses 
of  the  organism  floating  in  them.  On  potato  it  forms  a 


228  A  MANUAL  OF  BACTERIOLOGY 

growth  similar  to  that  on  agar.  When  grown  in  milk  it 
produces  coagulation.  Acid  production  (lactic  and  butyric 
acids)  can  be  demonstrated  by  growing  on  a  neutral  litmus 
glucose-agar.  When  grown  in  broth  or  peptone  water 
it  gives  the  indole  reaction  with  the  addition  of  a  nitrite, 
but  not  without. 

The  rate  of  liquefaction  of  gelatin  and  the  pigment 
production  vary  ;  the  latter  is  sometimes  much  deeper 
than  at  others,  recently  isolated  cultures  show  it  better 
than  old  ones,  and  the  presence  of  oxygen  also  seems 
to  be  necessary.  The  amount  of  acid  production  appears 
to  vary  directly  with  the  virulence,  which  is  likewise  very 
variable. 

Pathogenicity . — The  Micrococcus  pyogenes,  var.  aureus, 
is  by  far  the  commonest  of  all  organisms  met  with  in 
suppurative  processes.  Ogston  found  it  alone  in  thirty- 
four,  and  associated  with  the  Streptococcus  pyogenes  in 
sixteen,  out  of  sixty-four  cases  of  abscess.  It  occurs  in 
acute  abscesses,  boils,  and  acne,  in  some  cases  of  puer- 
peral fever  and  infective  endocarditis,  and  is  almost 
invariably  found  in  osteomyelitis,  but  only  occasionally 
in  pyaemia.  The  organism  injected  under  the  skin  of 
man  or  animals  produces  an  abscess,  and  injection  into 
the  blood-stream  under  certain  conditions  is  followed  by 
infective  endocarditis  or  pyaemia.  Impetigo  pustules  are 
produced  by  inunction  into  the  skin. 

It  may  be  said  to  be  universally  present  on  all  parts 
of  the  skin,  and  in  the  mouth,  and  is  frequently  met  with 
in  the  air.  According  to  Sternberg,  recent  cultures  in 
gelatin  are  destroyed  by  an  exposure  to  a  temperature  of 
56°  to  58°  C.  for  ten  minutes  ;  but  when  dried  much 
higher  temperatures,  90°  to  100°  C.,  are  required,  and  in 
the  dried  state  (on  a  cover-glass)  it  retains  its  vitality  for 
more  than  ten  days.  According  to  different  experimenters, 
from  five  to  fifteen  minutes  are  required  to  destroy  it 


PLATE  I. 


'.- 


Phagocytosis  by  polymorphonuclear  leucocytes,     a.  M.  pyogenes,  var. 
aureus.     b.  B.  tuberculosis. 


c.  M.  pyogenes,  var.  aureus  in  pus.     Smear 
preparation,      x  1000. 


d.  M.  pyogenes,  var  aureus. 

Gelatin  stab-culture, 

four  days  old. 


MICROCOCCUS  PYOGENES  229 

with  a  1-1000  mercuric  chloride  solution  ;  but  it  is  evident 
that  much  depends  on  the  state  of  aggregation  of  the 
organisms,  and  Abbott  has  shown  that  while  most  of  the 
cocci  in  a  culture  are  destroyed  in  five  minutes,  a  few  may 
survive  much  longer. 

Toxins. — In  a  case  of  infective  endocarditis  examined 
by  Sidney  Martin,  due  to  the  M.  pyogenes,  var.  aureus,  a 
large  amount  of  an  albumose  and  of  a  basic  body  was 
extracted  from  the  blood  and  spleen.  The  albumose 
produced  fever  and  wasting,  and  retarded  the  coagulation 
of  the  blood. 

Leber  extracted  a  crystalline  body,  which  he  termed 
phlogosin,  from  cultures  of  the  M .  pyogenes,  var.  aureus, 
and  Brieger  also  obtained  a  crystalline  base. 

The  decomposition  products  of  the  action  of  the  M. 
pyogenes,  var.  aureus,  on  egg-albumen  are,  according  to 
Emmerling,  phenol,  indole,  and  skatole,  many  volatile  and 
non- volatile  acids,  betaine,  and  trimethylamine. 

Anti-serum. — Attempts  have  been  made  to  prepare  an 
anti-serum  by  the  injection  of  cultures,  but  the  serum  is  of 
no  practical  value.  A  vaccine,  prepared  by  heating  a 
suspension  of  an  agar  culture  to  65°  C.  for  half  an  hour 
and  standardising,  has  been  used  with  much  success  in 
chronic  staphylococcic  infections,  such  as  acne  and  boils. 

Micrococcus  pyogenes,  var.  albus,  and  var. 
citreus.  Micrococcus  epidermidis.  Micro- 
coccus  cereus 

These  organisms  are  of  rarer  occurrence  than  the 
preceding  one.  In  morphology  and  cultural  characteristics 
the  first  two  agree  with  the  Micrococcus  pyogenes,  var. 
aureus,  except  that  the  albus  produces  a  white,  shining, 
porcelain-like  growth,  and  the  citreus  a  lemon-yellow 
growth,  on  agar.  They  are  said  to  be  less  pathogenic  than 


230 


A  MANUAL  OF  BACTERIOLOGY 


the  aureus,  and  are  only  occasionally  found  alone,  being 
usually  associated  with  the  aureus.  Cheyne,  however, 
states  that  in  his  experience  the  albus  is  more  virulent 
than  the  aureus,  and  mixed  infections  with  the  aureus  are 
regarded  as  more  severe  than  infection  with  the  aureus 
alone.  The  albus  has  been  found  in  some  cases  of  pan- 
ophthalmitis,  and  is  said  by  Fliigge  to  be  commoner  than 
the  aureus  in  the  lower  animals. 

Chief  Types  of  Human  Micrococci 


Acid  formation 

^ 

3 

"o   • 

"o 

from 

CO 

Organism. 

Broth 
culture. 

Pigment 
on  agar. 

ot  in  mil 

5  g 

eduction 
eutral  rei 

sduction 
nitrate. 

to 

1 

I 

i 

| 

1 

I 

'S 

G 

s 

C<  B 

P3 

*s 

1 

o 

(5 

Micrococcus 

Turbid 

Orange, 

+ 

+ 

0 

+ 

+ 

+ 

+ 

+ 

+ 

pyogenes 

yellow, 

or  white 

Micrococcus 

Turbid 

White 

+ 

+ 

_l_ 

+ 

+ 

+ 

+ 

0 

Feeble. 

epidermis 

Micrococcus 

Clear 

White 

0 

0 

0 

4 

+ 

0 

+ 

0 

0 

salivarius 

Scurf  micro- 

Turbid 

White 

0 

0 

0 

+ 

0 

0 

0 

+ 

0 

coccus 

or  clear 

Andrewes  and  Gordon1  regard  the  aureus,  albus,  and 
citreus  merely  as  variants  of  a  single  species,  the  Micro- 
coccus  pyogenes.  They  found  that  every  variety  of  colour, 
from  orange,  through  yellow  to  white,  might  be  obtained 
by  cultivation.  The  Micrococcus  fiavescens,  met  with  by 
Babes  in  abscesses,  may  probably  be  placed  in  the  same 
category.  On  the  other  hand,  the  Micrococcus  epidermidis 
(albus),  first  described  by  Welch  as  occurring  on  the  skin, 
in  stitch  abscesses,  etc.,  and  feebly  pathogenic  compared 

1  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1905-06,  p.  543. 


MICROCOCCUS  ZYMOGENES  231 

with  the  M.  aureus,  is  stated  by  these  authors  to  be 
perfectly  distinct  from  the  foregoing.  Other  organisms 
which  are  occasionally  met  with  in  abscesses,  the  Staphylo- 
coccus  cereus  albus  and  S.  cereus  flavus  of  Passet,  form 
shining  waxy  growths  on  agar,  and  do  not  liquefy  gelatin, 
and  are  probably  variants  of  another  species,  which  may 
be  termed  the  Micrococcus  cereus.  There  may  be  many 
other  varieties  of  micrococci  not  yet  properly  differentiated.1 
Well-defined  micrococci  occur  in  the  saliva  (M.  salivarius), 
and  in  the  scurf  from  the  scalp.  Andrewes  and  Gordon 
give  a  differential  Table  (see  p.  230)  of  some  of  these 
micrococci. 

Micrococcus  zymogenes 

Isolated  by  MacCallum  and  Hastings2  from  a  case  of 
acute  endocarditis.  A  minute  micrococcus,  non- motile, 
and  staining  by  Gram's  method.  On  surface  agar  it  forms 
a  thin,  slightly  elevated,  moist,  glistening,  greyish-white 
growth.  In  gelatin  stab-cultures  the  growth  is  somewhat 
opaque  and  granular,  with  slow  liquefaction.  Blood- 
serum  is  slowly  liquefied.  On  potato  a  thick,  moist, 
dirty-white  growth  develops,  becoming  dry  and  brownish 
after  three  days.  Broth  becomes  slightly  clouded  after 
twenty-four  hours'  growth,  but  in  three  to  four  days  the 
organisms  settle  to  the  bottom,  leaving  the  medium  clear. 
Neither  indole  nor  gas  is  formed.  In  neutral  litmus  milk 
the  litmus  is  decolorised  after  a  few  hours,  and  in  twenty- 
four  hours  the  milk  is  firmly  curdled.  Somewhat  later 
liquefaction  of  the  curd  ensues  from  above  downwards ; 
at  first  the  turbid  fluid  is  reddish  in  the  superficial  layer 
and  yellowish  below ;  ultimately  the  whole  curd  is  trans- 
formed into  a  turbid  liquid  with  a  reddish  colour  through- 
out. These  changes  in  milk  are  characteristic  of  the 

1  See  Gordon,  Rep.  Med.  Off.  LOG.  Gov.  Board  for  1903-04,  p.  388. 

2  Journ.  Exp.  Med.,  vol.  iv,  1899,  p.  521. 


232  A  MANUAL  OF  BACTERIOLOGY 

organism.  It  is  pathogenic  to  white  mice,  hardly  so  to 
guinea-pigs  and  white  rats,  and  moderately  so  to  rabbits ; 
intra-venous  inoculation  into  the  latter  sometimes  sets 
up  an  endocarditis.  Harris  and  Longcope1  have  reported 
five  more  instances  of  the  occurrence  of  this  organism 
(once  from  a  cesspool,  four  times  as  secondary  invasions 
at  autopsies),  and  Birge2  has  isolated  a  similar  but  less 
virulent  organism  from  the  larynx  of  crows.  Braxton 
Hicks3  has  also  isolated  this  organism  from  a  case  of 
malignant  endocarditis. 

Micrococcus  neoformans 

This  organism  was  isolated  by  Doyen  from  malignant 
growths,  and  was  supposed  by  him  to  be  the  causative 
organism  of  malignant  disease.  It  is  a  typical  Gram- 
positive  coccus,  giving  a  white  growth  on  agar  and 
liquefying  gelatin  in  three  to  four  days.  According  to 
Dudgeon  and  Dunkley,4  it  gives  all  Gordon's  fermenta- 
tion tests  for  the  M.  pyogenes,  var.  albus,  except  that  it 
does  not  acidify  mannitol. 

The  serum  of  patients  suffering  from  malignant  disease  does  not 
give  any  marked  agglutination  with  the  M.  neoformans,  nor  does 
it  contain  opsonins  specific  for  the  organism.  The  M .  neoformans 
is  non-pathogenic  for  rats  and  mice. 


The  Streptococci 

Many  streptococci  of  very  variable  virulence  occur  in 
man  and  animals.  Formerly  only  one  pathogenic  species 
was  described,  Streptococcus  pyogenes,  now  several  varieties, 
if  not  species,  are  recognised. 

1  Centr.  f.  Bakt.  (]>  Abt.),  vol.  xxx,  1901,  p.  353. 

2  Johns  Hopkins  Hosp.  Bull.,  vol.  xvi,  1905,  p.  309. 

3  Trans.  Eoy.  Soc.  Med.,  vol.  v,  1912,  Path.  Sect.,  p.  126. 
Journ.  of  Hygiene,  vol.  vii,  1907,  p.  13. 


PLATE  II. 


a.  Streptococcus  pyogenes  in  pus.     Smear  preparation,      x  1000. 


6.  Streptococcus  pycgenes.     Film  preparation 
of  a  broth  culture,      x   1500. 


c.  Streptococcus  pyogenes. 

Pure  culture  on  glycerin 

agar. 


STREPTOCOCCI  233 

Morphology. — The  streptococci  are  non-motile  cocci, 
each  cell  measuring  about  1  JUL  in  diameter.  They  stain 
well  with  anilin  dyes  and  are  Gram-positive. 

Fission  takes  place  in  one  direction  only,  so  that  chains 
of  cocci  are  formed.  A  cell  here  and  there  in  a  chain  is 
often  somewhat  larger  than  its  fellows,  and  some  authors 
have  considered  these  enlarged  individuals  to  be  arthro- 
spores. 

The  length  of  the  chains  is  very  variable  and  may  be 
modified  by  cultivation,  and  occasionally  branch- chains 
form. 

Von  Lingelsheim  distinguished  two  varieties,  brevis  and 
longus,  the  former  rendering  broth  turbid,  growing  in 
short  chains,  and  being  non-pathogenic  to  mice  and 
rabbits,  the  latter  leaving  the  broth  clear,  growing  in  long 
chains,  and  always  pathogenic  to  these  animals. 

Gordon1  divided  the  streptococci  into  four  varieties,  viz. 
(1)  the  S.  longus,  isolated  from  the  mouth,  restricted  to 
an  organism  forming  exceptionally  long  chains ;  (2)  S. 
medius,  including  the  majority  of  streptococci  from  pus, 
sepsis,  and  erysipelas,  and  Lingelsheim's  longus ;  (3)  S. 
brevis,  including  Lingelsheim's  brevis  and  the  Diplococcus 
pneumonia ;  (4)  S.  scarlatince  or  conglomerate,  isolated 
from  scarlatinal  angina. 

Cultural  reactions. — The  streptococci  can  be  cultivated 
on  the  ordinary  culture  media,  and  usually  grow  both 
aerobically  and  anaerobically.  On  agar,  or  better,  glycerin 
agar,  minute  whitish,  semi-transparent,  more  or  less 
isolated  colonies  form  in  twenty-four  to  forty-eight 
hours  (Plate  II.  c).  On  gelatin  the  growth  has  much  the 
same  characters,  and  is  better  seen,  as  this  medium  is 
clearer  than  agar,  but  it  takes  some  days  to  attain  the 
maximum.  In  stab-cultures  minute  spherical  colonies 
develop  all  down  the  line  of  the  stab,  but  without  invading 
1  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1898-99,  p.  482. 


234  A  MANUAL  OF  BACTERIOLOGY 

the  surrounding  medium ;  the  gelatin  is  not  liquefied. 
In  broth  a  flocculent  deposit  forms,  the  fluid  sometimes 
remaining  clear,  sometimes  becoming  turbid.  There  is 
no  growth  on  potato.  Litmus  milk  is  usually  acidified 
and  sometimes  coagulated,  and  acid  is  generally  produced 
from  glucose.  The  indole  reaction  can  be  obtained  in 
broth  cultures  in  seven  to  fourteen  days  on  the  addition 
of  a  nitrite,  but  not  without.  It  is  the  only  organism  with 
which  the  writer  is  acquainted  that  does  not  reduce  a  weak 
solution  of  methylene  blue. 

The  thermal  death-point  of  the  streptococci  is  53°  to 
55°  C.,  the  time  of  exposure  being  ten  minutes,  and  they 
are  destroyed  by  weak  solutions  of  disinfectants,  e.g. 
1-100  phenol,  in  ten  minutes. 

Considerable  attention  has  been  directed  to  the  dif- 
ferentiation of  streptococci  by  Houston,1  Andrewes,2 
Andrewes  and  Horder,3  Gordon,4  and  Besredka.  Con- 
siderable differences  are  found  in  the  fermentation  re- 
actions of  various  strains  of  streptococci,  and  Andrewes 
and  Horder  distinguish  (1)  Streptococcus  pyogenes  from 
pus,  erysipelas,  cellulitis,  pya3mia  and  septicaemia,  endo- 
carditis, etc.  (2)  S.  salivarius,  the  common  type  in  the 
saliva.  Also  met  with,  probably  as  a  "  terminal "  infec- 
tion, in  endocarditis  and  septicaemia.  Shades  into  the 
S.  fcecalis  and  S.  anginosus.  (3)  S.  anginosus,  from 
inflamed  and  scarlatina  throats,  endocarditis,  and  rheu- 
mati^m.  (4)  S.  fcecalis,  abundant  in  faeces,  air,  and  dust. 
Met  with  also  in  endocarditis,  meningitis,  cystitis,  and 
suppuration.  Two  strains  of  the  Diplococcus  rheumaticus 
proved  to  be  this  organism.  (5)  The  pneumococcus. 

1  Rep.  Ned.  Off.  Loc.  Oov.  Board  for  1902-03,  p.  511,  and  1903-04, 
p.  472. 

2  Lancet,  November  24,  1906. 

3  Ibid.  1906,  vol.  ii,  pp.  708,  775,  852. 

4  Ibid.  November  11,   1905,  and    Rep.   Med.  Off.  Loc.   Gov    Board 
for  1903-04,  p.  388. 


STREPTOCOCCUS  PYOGENES 


235 


(6)  S.  equinus,  present  in  the  intestine  of  herbivora.  They 
do  not  assert  that  these  are  absolutely  denned  species ; 
at  the  most  they  seem  to  be  species  in  the  making,  and  are 
connected  by  transitional  forms.  Walker1  does  not  con- 
sider that  these  reactions  afford  a  means  of  distinguishing 
definite  varieties  among  human  streptococci. 

Andrewes  and  Horder  give  the  following  Table  sum- 
marising the  characters  of  the  various  streptococci : 


r^ 

O-ri 

. 

>> 

^ 

. 

0 

• 

A 

i 

=' 

J 

q 

1 

.       0 

2  g 

1 

Name. 

H 

i 

0 

| 

£ 
~ 

= 
d 

"3 

1 

~ 

la 

1 

Is 

' 

1« 

a 

a 

V. 

. 

1 

°1 

i 

I2 

8 
B 

Streptococcus  pyogenes 
Streptococcus  salivarius 
Streptococcus  anginosus 
Streptococcus  fcecalis 
Streptococcus  equinus 
Streptococcus  pneumonice 

4- 
± 

± 

++++++  | 

-!- 

-;- 

.:. 

::: 

:; 

± 

r:: 
- 

-: 

5 

+-H-H++  1  | 

longus 
brevis 
longus 
brevis 
brevis 
brevis 

- 

0 
0 
0* 

+  =  Positive  or  acid-production.     —  =  Negative  or  no  acid-production. 

±  =  Acid-production  sometimes  present,  sometimes  absent. 

(These  differences  are  not  constant ;  with  various  strains  one  or  other  reaction 

may  be  lacking.) 

Crowe2  makes  use  of  Dorset's  egg- medium  with  the 
addition  of  0-005  per  cent,  of  neutral  red  for  the  purpose 
of  differentiating  streptococci. 

The  Streptococcus  pyogenes  is  found  in  some  16  per  cent, 
of  acute  circumscribed  abscesses.  It  is,  however,  especially 
frequent  in  spreading  inflammations,  lymphangitis,  cellu- 
litis,  and  progressive  gangrene,  and  is  a  common  cause  of 
septicaemia,  pyaemia,  and  puerperal  fever,  It  is  met  with 
in  about  one-third  of  the  cases  of  infective  endocarditis, 
occasionally  in  acute  osteomyelitis,  and  seems  to  be  the 
cause  of  the  septic  pneumonia  so  often  observed  after 
operations  about  the  mouth  and  throat. 

1  Proc.  Roy.  Soc.  Lond.,  B.  vol.  Ixxxiii,  1911,  p.  541. 
3  Proc.  Eoy.  Soc.  Med.,  vi,  1913  (Path.  Sec.),  p.  117. 


236  A  MANUAL  OF  BACTERIOLOGY 

A  streptococcus  (S.  viridans)  producing  a  green  growth 
on  blood-agar  and  belonging  to  the  S.  salivarius  group  has 
been  isolated  by  Major1  and  others  from  several  cases  of 
sub- acute  infective  endocarditis.  It  is  probably  not  a 
distinct  form  but  only  a  variant  of  the  S.  salivarius. 

In  erysipelas,  streptococci  are  present  in  the  lymphatics 
at  the  margin  of  the  zone  of  redness.  These  were  first 
isolated  by  Fehleisen,  who  described  the  organism  as  the 
Streptococcus  erysipelatis,  and  by  inoculation  experiments 
on  man  and  animals  demonstrated  its  causal  relation  to 
the  disease.  The  experiments  on  man  were  made  in  cases 
of  extensive  and  inoperable  carcinoma  and  sarcoma,  as  it 
had  been  noticed  that  malignant  tumours  were  frequently 
benefited  after  an  attack  of  erysipelas.  Several  cases  were 
inoculated,  and  in  all  but  one  typical  erysipelas  developed 
(see  Coley's  fluid,  p.  250).  Jordan,2  however,  produced 
typical  erysipelas  in  a  rabbit's  ear  not  only  with  the 
streptococcus,  but  also  with  staphylococci,  pneumococci, 
and  B.  coli,  and  although  human  erysipelas  is  generally 
caused  by  the  streptococcus,  this  disease  may,  therefore, 
occasionally  be  produced  by  staphylococci,  and  possibly 
by  the  pneumococcus,  B.  coli,  and  even  the  B.  typhosus. 

At  one  time  the  Streptococcus  erysipelatis  was  considered 
to  be  different  from  the  Streptococcus  pyogenes,  but  the 
two  organisms  are  now  regarded  as  identical,  the  differences 
in  cultural  characters  being  slight  and  not  constant.  A 
typical  erysipelas  in  the  human  subject  may  be  induced 
by  inoculation  with  a  pure  culture  of  a  streptococcus 
derived  from  a  case  of  suppurative  peritonitis,  and  an 
animal  immunised  against  a  streptococcus  derived  from 
a  case  of  erysipelas  is  also  immune  against  a  streptococcus 
isolated  from  an  abscess. 

The    different    effects    produced    by    the    Streptococcus 

1  Johns  Hopkins  Hosp.  Bull.,  xxii,  1912,  p.  326. 

2  Munch,  med.  Woch.,  August  27,  1901. 


ANTI-STREPTOCOCCIC  SERUM  237 

pyogenes,  abscess  in  one  case,  erysipelas  in  another,  cellu- 
litis  or  pyaemia  in  a  third,  are  attributable  partly  to  real 
differences  in  virulence,  partly  to  the  site  of  infection  and 
mode  of  entrance  into  the  body,  partly  to  real  differences 
existing  between  different  races  of  streptococci.  Strepto- 
cocci have  been  described  in  a  number  of  diseases  about 
which  we  know  little,  such  as  variola,  scarlatina  (S. 
scarlatince  or  conglomeratus),  and  vaccinia,  but  it  is  un- 
certain what  causal  relation  they  bear  to  these  conditions. 
Strangles,  a  disease  of  horses,  seems  to  be  due  to  strepto- 
cocci. 

Anti-serum. — The  important  lesions  due  to  the  strepto- 
coccus and  their  grave  nature  have  led  to  the  attempt  to 
prepare  an  anti-serum,  but  many  and  great  experimental 
difficulties  have  to  be  overcome  to  do  this.  The  virulence 
of  the  streptococcus  has  to  be  increased  by  passing  it 
through  a  series  of  rabbits,  and  it  is  only  by  growing  it  in 
serum  media  that  satisfactory  cultures  for  the  inoculation 
of  the  horses  can  be  prepared.  Human  serum  is  the  best, 
but  is  difficult  to  obtain ;  a  mixture  of  asses'  serum  and 
peptone  beef-broth  comes  next.  The  cultures  are  grown 
for  about  a  fortnight  and  are  then  inoculated  into  horses, 
first  killed  and  then  living  cultures  being  used,  and  after 
a  time  the  blood  acquires  anti-microbic  properties.  It 
is  customary  now  to  make  use  of  a  "  polyvalent "  serum, 
i.e.  one  prepared  by  the  injection  of  many  strains  of 
streptococci.  The  streptococcus  anti-serum  has  been 
employed  in  erysipelas,  cellulitis,  puerperal  fever,  and 
pyaemia,  in  many  cases  with  success.  Cheyne  suggested 
its  use  before  operations  about  the  mouth  and  throat  as 
a  preventive  of  septic  pneumonia,  but  a  vaccine  would 
probably  be  better  for  this  purpose. 

A  vaccine  prepared  by  sterilising  cultures  with  heat  has 
been  used  with  benefit  in  streptococcic  infections,  which 
do  not  run  too  rapid  a  course,  e.g.  infective  endocarditis. 


238  A  MANUAL  OF  BACTERIOLOGY 

Bacillus  pyocyaneus 

This  is  the  organism  found  in  green  and  blue  pus,  and  it 
also  occurs  on  the  surface  of  the  body.  Its  presence  in 
wounds  greatly  retards  healing,  and  occasionally  a  general 
toxaemia  may  result  from  it.  It  has  been  met  with  in 
otitis  media  and  in  the  green  pus  of  the  pleural  and  peri- 
cardial  cavities.  It  is  a  slender  bacillus  measuring  3  to 
4  jULt  frequently  united  in  pairs  and  forming  filaments.  It 
is  actively  motile,  does  not  form  spores,  and  is  aerobic 
and  facultatively  anaerobic.  It  does  not  stain  by  Gram's 
method.  On  gelatin  it  grows  freely  with  rapid  liquefaction, 
a  greenish,  fluorescent  colour  developing  in  the  liquid, 
while  whitish  flocculi  of  growth  sink  to  the  bottom.  On 
agar  a  whitish,  moist  layer  develops,  and  the  medium  is 
stained  a  greenish  colour.  On  potato  the  growth  is  a 
dirty  brown  or  sometimes  greenish. 

Milk  is  coagulated  and  a  greenish  colour  develops. 
Broth  becomes  turbid,  and  there  is  a  slight  film  formation 
with  a  greenish  colour.  Oxygen  is  necessary  for  the 
development  of  the  pigment,  which  is  generally  a  mixture 
of  a  blue  pigment,  pyocyanin,  and  a  yellow  one, 
pyoxanthose.  Pyocyanin  (C14H14N20)  is  said  to  be  an 
anthracine  derivative ;  it  is  soluble  in  chloroform,  and 
on  oxidation  yields  pyoxanthose.1  Various  races  of  the 
organism  exist,  differing  in  their  pigment  production. 

Subcutaneous  inoculations  of  a  small  amount  of  a  culture 
produce  local  abscesses  ;  larger  amounts  cause  oedema 
with  purulent  infiltration  of  the  tissues  and  death.  Animals 
can  be  vaccinated  by  means  of  small  quantities  of  living 
cultures  or  by  sterilised  cultures.  Sterilised  cultures  will 
prevent  infection  (experimentally)  by  anthrax  if  used 
early — that  is  to  say,  if  an  animal  be  inoculated  with 

:    1  See  Centr.  f.  BaU.,  xxv,  p.  897      Journ.  Exp.  Med.,  September- 
November  1899. 


BACILLUS  PYOCYANEUS  239 

anthrax,  and  shortly  afterwards  injected  with  a  broth 
culture  of  the  Bacillus  pyocyaneus,  a  fatal  result  is  averted. 
Emmerich  and  Loew1  claim  to  have  isolated  from  cultures 
a  ferment- like  body,  "  pyocyanase,"  which  they  state 
has  preventive  and  curative  properties  towards  anthrax 
and  diphtheria  infections.  Emmerich2  has  employed  the 
dry  pyocyanase  as  an  application  in  diphtheria  to  dissolve 
the  false  membrane. 

Williams  and  Cameron3  describe  four  cases  of  diarrhoea 
with  green  stools,  wasting  and  death  in  infants  in  which 
the  B.  pyocyaneus  was  obtained,  and  suggest  that  many 
cases  of  marasmus  may  be  due  to  it.  A  form  of  epidemic 
dysentery  seems  occasionally  to  be  caused  by  this  organism 
(see  "  Dysentery  ").  A  few  cases  of  general  infection  with 
this  organism  have  also  been  recorded.  It  has  also  been 
isolated  from  conditions  of  dermatitis  and  bullous  erup- 
tions.4 The  B.  pyocyaneus  has  been  found  in  water, 
dung,  soil,  and  in  the  effluent  from  filter  beds.  Lehmann  and 
Neumann  state  that,  with  the  exception  of  pathogenicity, 
there  is  no  essential  difference  between  this  organism  and  the 
B.fluorescens  liquefaciens  so  frequently  met  with  in  water. 

The  B.  pyocyaneus  seems  to  be  of  more  frequent  occur- 
rence and  of  greater  pathogenicity  in  the  tropics  than  in 
this  country.  A  disease  bearing  a  remarkable  similarity 
to  rabies  may  be  caused  by  it  (see  "  Kabies  "). 

Clinical  Examination 

In  many  cases  some  idea  can  probably  be  formed  as  to  the 
organisms  likely  to  be  present  in  the  pus  or  discharge,  etc.,  from 
the  clinical  characters  of  the  disease,  in  which  case  the  examination 
may  be  more  particularly  directed  towards  the  isolation  of  the 
suspected  organism.  For  example,  in  a  urethral  discharge  the 

1  Zeitschr.  /.  Hyg.,  1899 ;    Centr.  f.  Bakt.,  xxxi  (Originate],  p.  1. 

2  Munch,  med.  Woch.,  November  5  and  12,  1907. 

3  Journ.  Path,  and  Bact.,  vol.  iii,  1896,  p.  344  (Refs.). 

4  See  Fernet,  Brit.  Wed.  Journ..  vol.  ii.  1904,  p.  992. 


240  A  MANUAL  OF  BACTERIOLOGY 

gonococcus  will  be  especially  looked  for,  in  an  empyema  following 
pneumonia  the  Diplococcus  pneumonia,  and  in  a  tropical  abscess 
of  the  liver  the  Amoeba  coli.  In  all  cases  the  pus  or  discharge  should 
be  collected  with  aseptic  precautions  in  sterile  capillary  pipettes 
or  in  sterile  test-tubes  at  the  time  of  operation.  The  discharge  from 
opened  abscesses  and  from  wounds  is  liable  to  become  contaminated 
and  the  original  infection  to  be  masked.  In  septic  wounds  the 
infection  may  be  a  mixed  one. 

In  all  cases  the  examination  should  be  commenced  as  early  as 
possible. 

(1)  Make  several  smears  from  the  pus  or  discharge. 

(2)  Stain  one  or  two  of  these  with  L6 filer's  blue  and  one  or  two 
by  Gram's  method.     Mount  and  examine  microscopically. 

(a)  If   staphylococci   only   are    detected,    the   presence   of   the 
ordinary  pyogenic  cocci  may  be  suspected.    Proceed  as  in  3,  4,  and  5. 

(b)  If  encapsuled  diplococci  are  detected,  suspect  the  presence  of 
the  Diplococcus  pneumonice,  and  proceed  as  in  3,  5,  and  7. 

(c)  If  diplococci  and  tetracocci  are  present,  note  whether  they 
are  in  groups  within  the  pus-cells  ;   if  so,  suspect  the  presence  of 
either  the  gonococcus  or  Diplococcus  intracellularis    meningitidis, 
and  proceed  as  in  6. 

(d)  If  free  tetracocci  are  detected,  suspect  the  presence  of  the 
Micrococcus  tetragenus,  and  proceed  as  in  3  and  4  (rare). 

(e)  If    streptococci    are    present    the    Streptococcus    pyogenes   is 
probably  the  species.     Proceed  as  in  3,  4,  and  5. 

(/)  If  bacilli  are  present  they  may  be  the  colon  bacillus,  the 
Bacillus  Welchii  (aerogenes  capsulatus),  the  bacillus  of  malignant 
oedema,  the  tetanus  bacillus,  the  typhoid  bacillus,  the  Bacillus 
pyocyaneus,  or  putrefactive  bacilli  of  the  Proteus  group  (which  see). 
The  result  of  Gram-staining  and  the  clinical  history  of  the  case  will 
be  some  guide. 

a.  The  colon  bacillus,  especially  frequent  in  suppurative  peri- 
tonitis and  in  diseases  of  the  urinary  organs.  (See  p.  387). 

/3.  The  Bacillus  Welchii  (aerogenes  capsulatus},  especially  met  with 
in  foul  wounds  and  gangrenous  conditions,  with  much  development 
of  gas.  (See  Chapter  XIII.) 

y.  The  bacillus  of  malignant  oedema  occurs  in  septic  wounds 
with  septicsemic  complications.  (See  Chapter  XIII.) 

c).  The  tetanus  bacillus  is  found  in  the  wound  in  cases  of  traumatic 
tetanus.  (See  Chapter  XIII.) 

f.  The  typhoid  bacillus  is  rare  ;  it  may  occur  in  suppurative 
conditions  complicating  or  following  typhoid  fever.  Proceed  as  in 
3  and  4.  (See  also  p.  355.) 


MICROCOCCUS  MENINGITIDIS  241 

£.  When  the  Bacillus  pyocyaneus  is  present  the  pus  or  discharge 
may  be  blue.  Proceed  as  in  3  and  4. 

(g)  If  yellow  granules,  having  a  rosette-like  structure  micro- 
scopically, are  present,  actinomycosis  may  be  suspected  and 
examined  for  by  the  methods  given  in  Chapter  XV. 

(h)  If  thread  forms  be  present,  streptothrix  or  aspergillar  infection 
may  be  suspected  (see  Chapters  XV  and  XVII) :  if  large  round  or 
ovoid  cells  or  yeast -like  forms,  Blastomycetes  or  Sporotrichon 
(Chapter  XVI). 

(i)  If  a  mixture  of  organisms  be  present,  agar  and  gelatin  plate 
cultivations  should  be  prepared  and  further  examined  by  sub- 
cultures from  the  colonies. 

(j)  If  no  organisms  can  be  detected  microscopically,  proceed  as 
in  (3),  (7),  or  (9).  In  the  pus  of  ordinary  abscesses  micro-organisms 
can  generally  be  detected,  unless  caused  by  the  tubercle  or  glanders 
bacillus,  the  pneumococcus,  or  the  Amoeba  coli.  In  broken-down 
granulomata,  e.g.  gummata,  if  unopened,  no  organisms  may  be 
present. 

(3)  Make  several  cultivations  on  agar  and  gelatin  (anaerobic  if 
required),  and  examine  microscopically  and  by  subcultures  when 
the  growths  have  developed. 

(4)  Make  two  or  three  sets  of  agar  and  of  gelatine  plate  cultiva- 
tions.    Examine  the  colonies  microscopically  and  by  subcultures. 

(5)  Stain  two  or  three  of  the  cover-glass  preparations  by  Gram's 
method,  and  counter-stain  with  Bismarck  brown. 

(6)  The  gonococcus  and  Diplococcus  intracellularis  may  be  identi- 
fied and  distinguished  by  the  methods  detailed  at  pp.  247  and  242. 

(7)  Inoculate    guinea-pigs    or    mice   subcutaneously   and   intra- 
peritoneally  with  the  material. 

(8)  Organisms  can  rarely  be  detected  in  the  blood  by  a  micro- 
scopical examination  of  stained  films.     Therefore  2-5  c.c.  of  blood 
should  be  withdrawn  and  cultivated  (p.  126). 

(9)  If  the  abscess  be  probably  a  tropical  abscess  of  the  liver,  the 
pus  or  scrapings  from  the  wall  of  the  abscess  should  be  examined 
for  the  presence  of  the  Amoeba  coli.     (Chapter  XVIII.) 


Micrococcus  meningitidis 1 

Weichselbaum  in  1887  isolated  from  cases  of  epidemic 
cerebro-spinal  meningitis  (spotted  fever)  a  coccus  which 

1  See  Gordon,  Rep.  Loc.  Gov.  Board,   1907  (Bibliog.) ;    Arkwright, 
Journ.  of  Hygiene,  vol.  vii,  1907,  p.  193  ;    vol.  ix,  1909,  p.  104. 

16 


242  A  MANUAL  OF  BACTERIOLOGY 

he  named  the  Diplococcus  intracellularis  meningitidis,  and 
further  research  has  confirmed  the  accuracy  of  Weichsel- 
baum's  discovery  and  the  etiological  relationship  of  the 
organism  to  the  disease. 

Morphology,  etc. — The  meningococcus,  as  it  may  be 
termed,  occurs  as  single  cocci  and  diplococci  in  groups 
within  the  leucocytes  (Plate  III.  a)  ;  in  grouping  and 
general  appearance,  in  fact,  it  closely  resembles  the  gono- 
coccus,  and,  like  the  last-named,  is  Gram- negative,  though 
staining  well  with  the  ordinary  anilin  dyes  and  with  the 
Leishman  stain.  In  cultures  it  occurs  as  cocci,  diplococci, 
and  occasionally  as  tetrads. 

Cultural  characters. — The  meningococcus  is  an  obligatory 
aerobe,  and  does  not  grow  at  a  temperature  below  25°  C. 
It  will  occasionally  grow  in  primary  culture  on  glycerin 
agar,  but  frequently  not,  though  when  acclimatised  it 
grows  fairly  well  on  agar  and  in  broth.  The  organism 
develops  best  on  agar  smeared  with  blood,  on  ascitic-fluid 
agar  or  broth,  or  on  the  nutrose  ascitic  agar  of  Wassermann 
(termed  by  Gordon  "  nasgar  ")  : 

Ascitic  fluid  .  .  .  .  .16  c.c. 
Distilled  water  .  .  .  .  .35  c.c. 
Nutrose  ......  1  grm. 

The  mixture  is  placed  in  a  flask,  brought  to  the  boil  with  constant 
shaking,  and  filtered.  It  is  then  mixed  with  double  the  volume  of 
ordinary  nutrient  agar,  steamed  for  thirty  minutes,  filtered,  and 
filled  into  tubes. 

The  colonies  of  the  meningococcus  on  this  medium  after 
twenty-four  hours'  incubation  at  37°  C.  appear  as  moist, 
grey,  translucent,  circular  or  oval  discs  with  regular 
outline ;  after  a  further  twenty  hours'  growth  they  may 
attain  a  diameter  of  3  to  4  mm.  The  colonies  never 
exhibit  any  yellowish  coloration  as  do  those  of  some  other 
Gram-negative  cocci.  Ascitic  fluid  broth  (ascitic  fluid 
1  part,  broth  9  parts)  is  also  a  good  culture  medium,  and 


MICROCOCCUS  MENINGITIDIS  243 

it  grows  in  milk  without  clotting  or  change  in  reaction. 
Arkwright  found  that  grown  in  gelatin  at  37°  C.  the 
meningococcus  causes  liquefaction,  while  the  M.  catarrhalis 
does  not.  The  organism  needs  constant  transplantation 
to  maintain  vitality  in  culture.  The  fermentation  re- 
actions, which  are  somewhat  variable,  are  given  in  the 
table  on  p.  248. 

Symmers  and  Wilson1  examined  the  fermentation 
reactions  of  a  number  of  strains  of  the  meningococcus. 
Glucose,  maltose,  and  dextrin  were  fermented  with  the 
production  of  acid,  laevulose,  galactose,  lactose,  mannitol, 
dulcitol,  anda  number  of  glucosides  were  never  fermented. 

Pathogenesis. — In  man  the  organism  causes  epidemic 
cerebro- spinal  meningitis,  and  is  occasionally  met  with 
in  sporadic  cases  of  cerebro-spinal  meningitis.  It  is  also 
capable  of  producing  a  hsemorrhagic  septicaemia  without 
meningitis.  It  occurs  in  the  cerebro-spinal  fluid  (obtained 
by  lumbar  puncture)  in  the  blood  in  25  per  cent,  of  the 
cases  provided  quantities  of  5  to  20  c.c.  be  cultured, 
sometimes  in  the  upper  respiratory  passages,  particularly 
the  nose,  in  the  middle  ear,  eye  and  joints.  Park  states 
that  the  organism  is  usually  present  in  the  nose  in  the 
early  days  of  the  illness.  The  meningococcus  is  patho- 
genic to  mice  and  guinea-pigs  by  intraperitoneal  or  intra- 
pleural,  but  not  by  subcutaneous,  injection.  Intraspinal 
injection  into  monkeys  produces  a  typical  meningitis. 

An  agglutination  reaction  is  given  in  some  cases,  but 
is  neither  constant  nor  marked  enough  to  form  a  sure 
means  of  diagnosis. 

Symmers  and  Wilson1  have  found  that  the  blood  of 
epidemic  cerebro-spinal  meningitis  cases  may  occasionally 
agglutinate  the  B.  typliosus  and  B.  coli  in  comparatively 
high  dilutions. 

1  Journ.  of  Hygiene,  vol.  ix,  1909,  p.  9. 
1  Ibid.  vol.  viii,  1908,  p.  314. 


244  A  MANUAL  OF  BACTERIOLOGY 

Vaccine  and  anti-serum. — Cases  have  been  reported  of 
remarkable  benefit  derived  by  vaccinating  with  killed 
cultures. 

Flexner  has  prepared  an  anti- serum  with  which  suc- 
cessful results  have  been  obtained. 

Still  observed  in  simple  posterior  basic  meningitis  of  infants  a 
diplococcus  closely  resembling  the  meningococcus  but  growing  more 
freely  on  agar,  etc.  By  some  it  is  regarded  as  an  attenuated  form 
of  the  latter.  According  to  Arkwright  it  does  not  liquefy  gelatin, 
and  grows  on  this  medium  at  22°  C.,  fails  to  produce  acid  from 
glucose,  maltose,  and  galactose,  and  is  not  agglutinated  by  a 
meningococcus  serum.  It  is  in  these  respects  very  like  the 
M .  cinereus  of  Lingelsheim.  Wollstein  1  failed  to  find  any  reliable 
criteria  of  difference  between  strains  of  the  D.  intracellularis  and 
several  cultures  obtained  from  cases  of  posterior  basic  meningitis. 
Houston  and  Rankin  2  found  that  ten  Gram-negative  cocci  isolated 
from  cases  of  sporadic  cerebro -spinal  meningitis  differed  from  the 
D.  intracellularis  in  respect  of  their  opsonins  and  agglutinins, 
though  eight  of  them  were  identical  with  the  meningococcus  in  fer- 
mentative power.  Diplococcus  crassus  (Gram-positive),  D.  mucosus 
(grows  on  gelatin),  D.  flavus  (produces  yellow  pigment),  and 
M.  catarrhalis,  the  three  latter  Gram-negative,  may  occur  in  the 
naso-pharynx.  (See  Arkwright,  loc.  cit.,  also  p.  248.) 

Gram-positive  cocci  and  other  organisms  may  occasionally  cause 
a  sporadic  cerebro-spinal  meningitis,  e.g.  the  pneumococcus,  typhoid 
and  Gartner  bacilli,  and  streptococci  (S.fcecalis  and  S.  salivarius, 
Symmers  and  Wilson,  loc.  cit.  1909). 


Micrococcus  gonorrhoeas 

The  Micrococcus  gonorrhcece  was  discovered  by  Neisser  in 
1879  in  cases  of  gonorrhceal  urethritis.  In  gonorrhoeal 
pus  it  occurs  usually  in  pairs,  occasionally  in  tetrads,  the 
elements  of  which  are  somewhat  ovoid  in  shape,  their 
opposed  surfaces  being  flattened.  The  organism  has  a 
characteristic  arrangement :  it  occurs  in  groups  within 

1  Studies  from  the  Rockefeller  Inst.,  vol.  x,  1910,  No.  13. 

2  Brit.  Med.  Journ.,  1907,  vol.  ii,  p.  1414. 


PLATE   III. 


a.  The  meningococcus.      Smear  of  cerebro-spinal  fluid.      x   1000. 


b.  The  gonococcus.      Smear  of  gonorrhoaa    pus.      x    1500. 


MICROCOCCUS  GONORRHCE^E  245 

the  pus-cells  (Plate  III.  &).  The  individual  cocci  vary 
somewhat  in  size,  the  average  being  about  0*7  //,  in  the 
long  and  0-5  JUL  in  the  short  diameter.  It  stains  readily 
with  the  ordinary  anilin  dyes,  Loffler's  blue  being  perhaps 
the  best,  but  is  decolorised  by  Gram's  method — an  im- 
portant practical  distinction  from  many  other  cocci. 

Cultural  characters. — The  gonococcus  is  difficult  to 
cultivate,  and  usually  soon  dies  out  under  cultivation — 
within  a  week,  unless  transferred  to  fresh  soil — but  it 
does  not  seem  to  lose  its  virulence.  Growth  takes  place 
between  25°  and  38°  C.,  but  the  optimum  temperature 
is  between  35°  and  37°  C.  It  is  aerobic,  and  possibly 
facultatively  anaerobic,  and  will  develop  on  a  feebly 
alkaline  or  acid  soil.  The  ordinary  agar  and  gelatin 
media  are  useless  for  the  cultivation  of  the  gonococcus ; 
it  will  grow  only  on  a  medium  containing  "  native " 
protein.  Blood- serum  agar  gives  fair  results,  but  the 
ordinary  Loffler's  blood-serum  is  of  no  use.  The  best 
medium  is  agar  smeared  with  blood.  Ordinary  sloping 
agar  tubes  or  small  agar  plates  may  be  employed.  Blood 
obtained  by  pricking  the  finger,  with  antiseptic  precautions, 
is  taken  up  in  a  sterile  capillary  tube  and  deposited  on 
the  agar.  A  trace  of  gonorrhoeal  pus,  collected  with 
aseptic  precautions,  is  taken  up  on  a  small  sterile  camel's- 
hair  brush,  and  is  rubbed  up  with  the  drop  of  blood  and 
smeared  over  the  surface  of  the  agar.  The  cultures  are 
incubated  at  37°  C.,  and  in  twenty-four  hours  the  colonies 
of  the  gonococci  appear  as  transparent  greyish  specks, 
which  increase  in  size  up  to  the  end  of  three  days.  At  this 
stage  the  colony  measures  1  to  2  mm.  in  diameter,  is 
raised,  brownish,  and  finely  granular  in  appearance,  and 
roundish  with  a  crinkled  margin.  The  cocci  from  cultures 
resemble  those  in  the  pus,  but  tetrads  are  more  frequently 
met  with.  Egg-broth  also  gives  good  results.  The  fer- 
mentation reactions  and  comparison  with  other  Gram- 


246  A  MANUAL  OF  BACTERIOLOGY 

negative  cocci  will  be  found  in  the  Table,  p.  248.  The 
specific  virulence  of  gonorrhoeal  pus  is  destroyed  by 
exposure  to  a  temperature  of  60°  C.  for  ten  minutes. 

Paihogenicity . — The  gonococcus  is  a  strictly  parasitic 
organism,  and  seems  exclusively  to  attack  man.  From 
inoculation  experiments  on  the  human  subjects  it  appears 
to  be  the  specific  organism  of  gonorrhoeal  urethritis  and 
vulvitis.  In  the  female  it  is  most  frequent  in  the  urethral 
or  vulvar  discharge,  less  so  in  that  from  the  cervical  canal, 
and  is  rarely  or  never  seen  in  a  purely  vaginal  one.  It  is 
generally,  even  at  an  early  stage,  associated  with  other 
organisms,  particularly  other  diplococci  (see  Table,  p.  248) 
which  have  to  be  distinguished  from  the  gonococcus. 
The  features  which  serve  to  identify  the  latter  are  its 
shape  and  size,  its  non-staining  by  Gram's  method,  its 
arrangement  in  groups  within  the  pus- cells,  absence  of 
growth  on  ordinary  media,  the  characters  of  the  colonies, 
and  the  fermentation  reactions. 

The  gonococcus  is  associated  with  a  variety  of  lesions 
besides  those  already  mentioned,  viz.  epididymitis, 
ovaritis,  salpingitis,  cystitis,  peritonitis,  arthritis,  and 
conjunctivitis.  It  has  been  met  with  in  the  blood,  and 
occasionally  produces  endocarditis,  pericarditis,  and 
meningitis.  The  gonococcus  is  fatal  to  guinea-pigs  and 
mice  by  intraperitoneal  inoculation. 

Toxin,  anti-serum,  and  vaccine. — Christmas1  found  that 
the  blood- serum  of  the  rabbit,  fluid  or  coagulated,  is  an 
excellent  culture  medium  for  the  gonococcus.  By  culti- 
vating the  gonococcus  for  ten  days  in  an  ascitic  bouillon 
mixture  he  succeeded  in  obtaining  a  toxin  which,  when 
injected  intravenously  into  rabbits  in  large  doses,  caused 
death,  in  smaller  doses  fever  and  loss  of  weight,  while 
precipitated  with  alcohol  and  injected  into  the  anterior 
chamber  of  the  eye  it  produced  severe  inflammation.  By 

1  Ann.  de  Vlnst.  Pasteur,  xi,  1897,  p.  609. 


MICROCOCCUS  GONORRHOLE  247 

injecting  rabbits  with  small  doses  of  the  toxin  immunisa- 
tion was  produced,  and  the  blood  acquired  antitoxic 
properties.  A  vaccine  may  be  prepared  by  sterilising 
cultures  with  heat,  and  has  proved  of  service  in  chronic 
gonorrhoeal  infections. 

Clinical  Diagnosis 

The  diagnosis  of  gonorrhoea  is  very  important,  not  only  in  clinical 
but  also  in  medico-legal  cases.  For  this  purpose  microscopical 
examination  and  culture  methods  are  made  use  of.  In  a  chronic 
gleet  the  material  must  be  examined  carefully  and  repeatedly. 

(1)  Microscopical  examination. — Several  thin  smear  specimens  of 
the  pus  or  discharge  should  be  prepared.     If  the  best  results  are 
desired  the  films  should  be  air-dried,  and  then  fixed  by  placing  in  a 
mixture  of  equal  parts  of  alcohol  and  ether  for  fifteen  minutes. 
After  fixing,  a  couple  of  the  films  are  stained  in  Loffler's  blue  for 
five    to    ten    minutes,    washed   in   water,    dried    and    mounted. 
Leishman's  stain  also  gives  good  results,  the  films  being  merely 
air-dried  and  not  fixed.     The  preparations  are  then  examined  with 
a  TV-inch  oil-immersion  ;  a  lower  power  lens  is  useless.     The  ovoid 
cocci  in  pairs,  and  occasionally  in  tetrads,  occurring  within  the 
pus-cells  in  groups  of  not  less  than  four  pairs  are  very  characteristic. 
Diplococci  situated  outside  the  pus-cells  should  be  neglected  (it  is 
to  be  noted  that  the  nuclei  of  the  pus-cells  are  deeply,  the  cytoplasm 
only  faintly,  stained  with  methylene  blue).     The  next  step  is  to 
ascertain  the  staining  reaction  by  Gram's  method.     Stain  two  more 
films  for  fifteen  minutes  in  anilin  gentian  violet,  dip  in  water,  place 
in  Gram's  iodine  solution  for  two  minutes,  decolorise  in  absolute 
alcohol  until  the  drainings  fail  to  stain  white  filter  paper,  and 
counter-stain  for  forty-five  seconds  in  a  saturated  aqueous  solution 
of    Bismarck    brown    diluted   with    three   times    its    volume    of 
distilled  water.     The  gonococci  are  decolorised,  and  take  up  the 
brown  stain.     In  chronic  urethritis  the  urine  may  be  centrifuged, 
and  preparations   are   made  from  the  deposit  and  threads   and 
stained. 

(2)  Culture  methods. — Whenever  a  diagnosis  is  of  great  importance 
an   attempt   should   be   made   to   cultivate   the   organism.     Plate 
cultures  of  agar  smeared  with  blood  as  described  (p.  245)  and  another 
set  with  agar  only  should  be  prepared  and  incubated  at  37°  C.     In 
forty-eight  hours  colonies  of  the  gonococcus  should  be  recognisable 
on  the  blood-agar,  but  not  on  the  plain  agar. 


248 


A  MANUAL  OF  BACTERIOLOGY 


If  cultures  are  obtained,  the  fermentation  tests  (see  below)  may 
be  applied. 

N.B.  The  greatest  caution  must  be  exercised  in  declaring  a  case 
free  from  infection  on  the  ground  of  NEGATIVE  results  of  the  examina- 
tion. 

The  Characters  of  the  Chief  Gram-negative  Cocci  (Gordon) 


Growth  on 

Growth  on 

0 

i 

0 
0 

I 

Organism  or  source. 

nutrose  ascitic 

gelatin  at 

Pathogenicity. 

8 

c* 

£ 

c3 

agar  at  37°  C. 

20°  C. 

0 

1? 

C3 

o 

0 

M.  catarrhalis.   Nasal 

Opaque, 

Positive  (grows 

Mice  and 

0 

0 

0 

0 

and  pharyngeal  dis- 

granular 

on  ordinary 

guinea-pigs  by 

charge 

agar  at  37°  C.) 

intraperitoneal 

inoculation 

only 

M.  intracellularis 

Clear,  smooth 

Negative 

In  some  cases 

4- 

-|- 

4- 

0 

(meningococcus)  . 

mice  and 

Cerebro-spinal  menin- 

guinea-pigs by 

gitis 

intraperitoneal 

inoculation 

only 

M.  gonorrhoea  (gono- 

No  growth 

Negative 

Ib. 

_l- 

-f- 

0 

0 

coccus).     Urethral 

unless  blood 

discharge 

added 

From  nasal  discharge 

Clear,  smooth, 

Negative  at 

Mice  and 

_l_ 

0 

_l_ 

0 

from  Hertford  case 
of  influenza-like  epi- 

later becomes 
yellowish 

first,  positive 
later  (grows  on 

guinea-pigs  by 
intraperitoneal 

demic  (see  "  Influ- 
enza ") 

ordinary  agar 
at  37°  C.) 

inoculation 

Ib.          .         . 

Opaque,  granu- 

Negative 

Ib. 

+ 

-(- 

+ 

+ 

lar 

From  urethra 

Opaque,  some- 

Positive 

— 

-f 

-J- 

+ 

-f 

what  granular, 

smooth  edges 

M.  melitensis.  Malta 

Creamy  and 

Positive 

Monkeys.    Also 

_ 

0 

0 

0 

fever 

slightly 

rabbits  and 

yellowish 

guinea-pigs  by 

intracorcbral 

inoculation 

+  =  acid. 


=  alkali. 


no  action. 


Micrococcus  catarrhalis  1 

This  organism  occurs  in  the  nose  and  throat  in  cases  of  catarrh, 
and  particularly  in  the  "  influenzal  cold  "  (see  "  Influenza  "),  in 
bronchial  catarrh,  and  occasionally  in  other  conditions  and  in  well 
people.  Morphologically  it  occurs  in  pairs  and  tetrads,  often 

1  See  Gordon,  Brit.  Med.  Journ.,  1905,  vol.  ii,  p.  423  ;  Arkwright, 
Journ.  of  Hygiene,  vol.  vii,  1907,  p.  145. 


MICROCOCCUS  CATARRHALIS  249 

within  the  polymorphonuclear  leucocytes.  It  is  Gram-negative. 
The  primary  generation  develops  feebly  on  agar,  but  subsequent 
generations  grow  fairly  well,  forming  whitish  translucent  colonies. 
Blood  or  ascitic  media  should  be  used  for  isolation.  Some  of  the 
fermentation  reactions  and  a  comparison  with  other  Gram-negative 
cocci  are  given  in  the  table  on  page  248. 


Micrococcus  tetragenus 

This  organism  is  frequently  met  with  in  phthisical  cavities  and 
may  be  expectorated  in  the  sputum,  and  has  also  been  found  in 
the  pus  of  acute  abscesses.  The  cells  occur  singly  (diameter  1  /*), 
in  pairs,  or  in  fours,  and  are  enclosed  within  a  capsule.  It  stains 
with  the  ordinary  anilin  dyes  and  also  by  Gram's  method.  On 
gelatin  it  develops  slowly,  with  the  formation  of  a  thick,  white, 
shining  growth  without  liquefaction.  On  agar  the  growth  has  much 
the  same  characters,  and  on  potato  is  white  and  viscous.  Inoculated 
into  animals,  particularly  mice,  a  local  abscess  may  form,  but  usually 
a  fatal  general  infection  ensues,  and  the  organism  is  found  in  the 
blood  and  organs. 

A  few  cases  of  general  infection  in  man  have  been  described. 


Sarcina  ventriculi 

An  organism  occurring  in  the  contents  of  the  stomach,  especially 
in  cases  of  dilated  stomach.  Originally  described  by  Goodsir  in 
1842. 

It  occurs  as  a  large  ovoid  cell,  several  of  which  are  grouped 
together  quadrilaterally  so  as  to  form  more  or  less  cubical  masses, 
the  so-called  "  woolpacks."  According  to  Falkenheim,  it  forms  on 
gelatin  in  thirty-six  to  forty-eight  hours  roundish,  prominent 
colonies  of  a  yellowish  colour,  and  in  neutral  hay  infusion  a  brownish 
film  and  flocculi.  It  produces  an  acid  reaction. 

Other  sarcinse  also  occur  in  the  stomach. 

Clinical  examination. — 1.  The  organism  can  be  detected  in  the 
vomit,  etc.,  most  readily  by  examination  in  the  fresh  state,  a  little 
of  the  material  being  placed  on  a  slide,  diluted  with  water  if  neces- 
sary, irrigated  or  not  with  iodine  solution,  covered  with  a  cover- 
glass,  and  examined. 

2.  Film  preparations  may  be  stained  with  weak  carbol  fuchsin, 
or  by  Gram's  method. 


250  A  MANUAL  OF  BACTERIOLOGY 

Other  Organisms  met  with  in  Suppurative 
and  Septic  Conditions 

Many  other  organisms  may  be  met  with  in  various  suppurative 
and  septic  processes,  e.g. : 

a.  The  B.  coli  in  cystitis  and  pyelitis,  ischio-rectal  abscess, 
peritonitis  associated  with  perforation  and  intestinal  obstruction, 
and  puerperal  fever  (see  Chapter  X). 

&.  The  Diplococcus  pneumonia  in  abscesses,  empyema,  arthritis, 
meningitis,  pericarditis,  peritonitis,  etc.  (see  Chapter  XII). 

c.  The  B.  typhosus   in   abscesses,  cholecystitis,  empyema,  and 
osteomyelitis  (see  Chapter  X). 

d.  The  B.  cedematis  and  B.  Welchii  in  foul,  gangrenous  wounds 
(see  Chapter  XIII). 

e.  The  B.  tuberculosis  and  B.  mallei  (see  Chapter  IX). 

/.  The  actinomyces  and  streptothrix  forms  (see  Chapter  XV). 
g.  Blastomycetes,   Sporotrichon  (see  Chapter  XVI)  and  Hypho- 
mycetes  (see  Chapter  XVII). 

h.  The  Amoeba  coli  (see  Chapter  XVIII). 
».  Capsulated  bacilli  (see  note,  p.  258). 

Coley's  Fluid 

This  preparation  consists  of  the  toxins  of  the  streptococcus  of 
erysipelas  and  the  B.  prodigiosus.  It  was  devised  by  W.  B.  Coley, 
of  New  York,  as  a  cure  for  inoperable  malignant  tumours,  particu- 
larly sarcoma.  The  treatment  is  based  on  the  undoubted  fact  that 
malignant  growths  may  decrease  or  even  disappear  completely 
after  an  attack  of  erysipelas  (p.  236).  Originally  prepared  by  grow- 
ing a  virulent  streptococcus  obtained  from  a  fatal  case  of  erysipelas 
in  bouillon  for  about  ten  days  ;  the  culture  is  then  inoculated 
with  the  B.  prodigiosus  and  the  two  are  allowed  to  grow  together 
for  another  week  or  ten  days.  The  culture  is  finally  heated  to  from 
58°  to  60°  C.  for  one  hour,  and  a  piece  of  thymol  added  to  preserve 
it.  The  fluid  is  now  prepared  by  growing  the  organisms  separately 
and  then  mixing  the  two  sterilised  cultures  in  proper  proportions. 

The  fluid  is  injected  subcutaneously  in  the  vicinity  of  the  tumour. 
The  primary  dose  recommended  is  J  minim  of  the  fluid.  The 
dose  is  gradually  increased  each  day  until  there  is  a  temperature 
reaction  of  103°  to  104°  F. 

Full  particulars  will  be  found  in  Coley's  paper  (Proc.  Roy.  Soc. 
Med.,  vol.  iii,  1909-10,  Surg.  Sect.,  p.  1). 


CHAPTER  VII 
ANTHRAX 

ANTHKAX  is  essentially  a  disease  of  cattle  known  as  splenic 
fever,  and  though  occurring  in  England  only  sporadically, 
or  in  small  outbreaks,  in  some  parts  of  the  world  it  assumes 
serious  proportions — as  in  Siberia,  where  it  has  been 
termed  the  Siberian  plague.  In  France  also  at  one 
time  it  ravaged  the  sheep  to  such  an  extent  as  to 
threaten  them  with  extinction.  Man  is  also  occasionally 
attacked. 

Anthrax  was  the  first  disease  to  be  definitely  associated 
with  a  specific  micro-parasite,  for  the  organism  was 
observed  as  glassy  homogeneous  rods  and  filaments  in  the 
blood  of  infected  animals  so  long  ago  as  1849  by  Pollender 
and  1850  by  Davaine,  and  the  latter  also  claimed  in  1863 
to  have  demonstrated  by  inoculation  experiments  the 
causal  relation  of  the  organism  to  the  disease.  Davaine's 
experiments  were  made  by  inoculating  an  animal  directly 
with  the  blood  from  an  infected  animal,  and  were,  there- 
fore, hardly  conclusive,  as  they  did  not  comply  with  the 
second  and  third  of  Koch's  postulates,  which  declare  that 
the  micro-organism  must  be  cultivated  outside  the  body, 
and  the  cultivated  organism  must  produce  the  disease  on 
inoculation,  and  the  objection  was  raised  that  infection 
was  due,  not  to  the  bacillus,  but  to  something  else  in  the 
blood.  This  objection  was  subsequently  removed  by  the 
work  of  Pasteur  and  of  Koch,  who  obtained  pure  cultures 
of  the  organism,  the  Bacillus  anihracis,  and  with  these 

251 


252  A  MANUAL  OF  BACTERIOLOGY 

produced  results  the  same  as  had  previously  been  obtained 
by  inoculation  with  the  blood  of  an  infected  animal. 

Morphology. — The  Bacillus  anihmcis  is  a  rod-shaped 
organism  varying  slightly  in  size  in  different  animals  and 
under  cultivation  ;  in  the  blood  it  measures  from  5  to  20  //. 
in  length  and  1  to  1-25  /x  breadth  (Plate  IV.  a),  but  in 
cultures  long  filaments  develop.  Examined  in  the  fresh 
and  living  condition  in  a  hanging- drop  preparation,  these 
rods  and  filaments  appear  homogeneous  or  slightly  granular ; 
in  stained  preparations,  however,  they  are  seen  to  be  made 
up  of  a  series  of  segments  with  unstained  interspaces, 
each  segment  measuring  about  4  to  5  /x  in  length,  and  the 
ends  of  the  segments  appear  cut  off  square,  provided  care 
has  been  taken  not  to  overheat  in  fixing  and  to  stain  with 
an  aqueous  solution ;  they  also  appear  to  be  encapsuled 
(p.  263).  In  the  blood  the  filaments  never  exceed  about 
five  or  six  segments  in  length,  except  perhaps  in  swine,  in 
which  animals  they  may  be  somewhat  longer.  In  cultures, 
however,  the  filaments  may  be  of  almost  unlimited  length, 
and  lie  parallel  to  one  another  or  in  more  or  less  tangled 
masses.  In  the  animal  body  during  life,  and  for  some 
hours  after  death,  spores  never  occur ;  but  in  cultures 
more  than  a  day  or  so  old,  and  from  which  oxygen  has  not 
been  excluded,  they  are  always  present,  almost  every 
segment  containing  one.  The  spores  are  ellipsoidal, 
measuring  about  1  ^  by  1-25  /x,  and  are  centrally  placed 
in  each  segment,  the  long  axis  corresponding  with  the 
long  axis  of  the  segment. 

Cultural  reactions. — The  anthrax  bacillus  is  aerobic  and 
facultatively  anaerobic ;  it  is  non-motile,  and  stains  well 
with  the  ordinary  anilin  dyes,  and  especially  so  by  Gram's 
method.  It  grows  readily  on  all  culture  media  at  from 
20°  to  37°  C.,  the  latter  being  the  optimum.  Develop- 
ment ceases  at  temperatures  below  about  15°  and  above 
5°  C.  Small,  cream-coloured,  granular  colonies  develop 


PLATE  IV. 


'/          V 


i£l 

>mli 


a.  Bacillus  anthracis.     Smear  of  blood  of  inoculated  guinea-pig. 
X    750. 


W  '  I 


6.  Anthrax.     Section  of  kidney  through  glomerulus.      x  500. 


ANTHRAX 


253 


in  a  gelatin  plate  in  about  thirty  hours,  and  in  two  to 
three  days  appear  as  small,  roundish,  cream-coloured 
pasty  masses  in  little  pits  in  the  gelatin,  due  to  its  lique- 
faction. Microscopically  the  colonies  are  somewhat  char- 
acteristic ;  each  consists  of  a  mass  of  wavy,  tangled 
filaments  like  a  tiny  wad  of 
cotton- wool.  In  gelatin  streak- 
cultures  development  is  slow, 
and  in  four  or  five  days  a 
creamy,  pasty  growth  forms 
in  a  trough  of  liquefaction.  In 
a  gelatin  stab- culture  (prefer- 
ably 5  per  cent,  gelatin)  lateral 
branches  spread  from  the  cen- 
tral growth,  longer  in  the  upper 
layers,  shorter  below,  so  that 
at  the  end  of  a  week  the  cul- 
ture is  like  an  inverted  fir  tree 
(Fig.  36),  and  the  gelatin  be- 
comes gradually  liquefied  from 
above  downwards.  The  colonies 
on  an  agar  plate  develop  in 
twenty  hours  at  37°  C.  as  cream- 
coloured  points.  The  surface 
colonies  microscopically  consist 
of  little  masses  of  wavy,  tangled 
filaments  (Plate  V.  a  and  b) ; 
"  they  are  not  circular  but  run  to  a  point  in  two  or  three 
directions,  with  gracefully  curved  margins  "  (Reichel),  and 
the  growth  is  sticky.  The  young  deep  agar  colonies,  which 
Eurich  x  considers  most  characteristic,  consist  of  interlacing 
knotted  coils  of  fine  filaments.  On  an  agar  surface  culture 
at  37°  C.  there  is  a  copious  development  in  eighteen  hours 
of  a  thick,  cream-coloured,  slimy  growth,  which  at  this 
1  Journ.  Path,  and  Bact.,  xvii,  1912,  p.  249. 


FIG.  36.— Anthrax.  Gelatin 
stab-culture.  Seven  days 
old. 


254  A  MANUAL  OF  BACTERIOLOGY 

early  stage  has  a  finely  granular,  ground-glass  appearance. 
On  blood-serum  a  thick  creamy  layer  forms,  with  slow 
liquefaction  of  the  medium.  On  potato  the  organism 
grows  freely  as  a  dry  greyish  layer,  with  an  abundant 
formation  of  spores.  In  broth  it  forms  a  somewhat  scanty 
flocculent  deposit,  the  broth  remaining  clear  and  giving 
the  indole  reaction. 

In  old  cultures  various  involution  forms  are  met  with ; 
the  rods  lose  their  regular  shape  and  become  swollen, 
producing  the  so-called  torula  forms,  while  the  homo- 
geneous appearance  of  the  protoplasm  changes  and 
becomes  granular.  Ultra-violet  rays  are  stated  by  Mme. 
Henri  to  produce  marked  mutations  of  the  anthrax 
bacillus  (see  p.  6).  Spores  are  found  in  all  culture  media 
when  there  has  been  free  access  of  oxygen,  as  in  surface 
cultures  on  potato  and  agar  ;  but  in  a  deep  broth  culture, 
where  the  supply  is  limited,  spore-formation  is  absent 
or  very  scanty.  Spores  are  never  met  with  in  the  living 
animal ;  they  only  appear  some  hours  after  death,  or 
when  matter  containing  the  bacilli  comes  in  contact  with 
air,  as  in  the  bloody  discharge  from  the  nostrils.  It  has 
therefore  been  supposed  that  oxygen  is  necessary  for 
spore- formation  to  take  place,  but  this  does  not  seem  to 
be  the  whole  explanation,  for  spores  form  in  an  atmosphere 
of  nitrogen,  though  they  do  not  do  so  in  one  of  hydrogen. 
The  life-history  of  the  organism  and  the  development  of 
spores  can  be  well  watched  in  a  hanging- drop  specimen 
prepared  by  inoculating  a  droplet  of  broth  with  the  blood 
of  an  infected  animal.  The  preparation  can  be  observed 
on  a  warm  stage,  or  examined  at  stated  times,  being  kept 
in  the  intervals  in  the  blood-heat  incubator.  At  the  end 
of  twenty-four  hours  the  short  filaments,  which  alone  are 
present  in  the  blood,  will  have  grown  so  long  that  they 
stretch  across  the  field,  while  the  protoplasm  has  become 
granular,  and  minute  shining  points  are  visible  here  and 
there.  In  another  twenty-four  hours  the  filaments  extend, 


PLATE  V. 


a.  Bacillus  anthracis.     Impression  preparation  of  a  surface  colony. 

X  40. 


b.  Bacillus  anthracis.     Impression  preparation  of  a  surface  colony. 
X   750. 


ANTHRAX  255 

the  protoplasm  becomes  still  more  granular,  and  the 
shining  spots  are  now  well-marked  ovoid,  highly  refractile 
bodies — the  mature  spores.  In  old  cultures  the  rods  and 
filaments  almost  disappear,  numbers  of  spores  alone 
remaining.  These  spores,  when  placed  under  favourable 
conditions  of  moisture,  warmth,  and  nutriment,  again 
produce  rods  and  filaments  ;  a  little  bud  appears  at  the 
extremity  of  the  long  diameter,  which  grows  in  length  and 
ultimately  becomes  a  mature  rod,  often  with  the  empty 
spore  capsule  embracing  one  end.  Sporeless  varieties  of 
the  anthrax  bacillus  have  been  •  obtained  by  cultivating 
under  unfavourable  conditions,  as  at  a  high  temperature 
(44°  C.)  or  in  the  presence  of  minute  quantities  of  anti- 
septics (1  :  1000  carbolic  acid). 

The  spores  are  of  considerable  practical  importance, 
for  they  are  highly  resistant  forms,  requiring  at  least  some 
minutes'  boiling  and  three  hours  in  dry  air  at  140°  C.  for 
their  destruction,  whereas  the  bacilli  without  spores  are 
destroyed  in  ten  minutes  in  the  moist  condition  by  a 
temperature  of  54°  C.  The  same  resistance  occurs  towards 
various  germicidal  substances.  While  1  per  cent,  carbolic 
acid  solution  quickly  destroys  bacilli  without  spores,  the 
spores  resist  5  per  cent,  carbolic  for  days,  and  at  least 
5  per  cent,  solutions  of  high-coefficient  phenoloid  dis- 
infectants, acting  for  not  less  than  twenty-four  hours 
at  20°  C.,  are  required  to  kill  the  spores.  The  resistance 
of  the  spores  is  stated  to  increase  with  their  age,  but  the 
writer  has  not  found  this  always  to  be  the  case.  Formalin 
and  a  formalin- containing  disinfectant,  "  Bacterol,"  seem 
to  have  a  selective  action  on  anthrax  spores  and  are 
efficient  disinfecting  agents  for  them.  Reichel  and  Gegen- 
bauer  recommend  for  the  purpose  a  mixture  of  10  per 
cent,  salt  and  1  per  cent,  hydrochloric  acid  at  30°  C., 
acting  for  twenty- four  hours.  Anthrax  spores  retain  their 
vitality  and  pathogenic  power  unimpaired  for  years  in  a 
dried  condition. 


256  A  MANUAL  OF  BACTERIOLOGY 

Certain  anthrax-like  bacilli  have  been  described  and 
have  to  be  distinguished  from  B.  anthracis,  e.g.  B.  pseudo- 
anthracis,  B.  anthracoides,  B.  anthracis  similis.  These  are 
non-pathogenic  and  are  haemolytic  for  rabbit,  sheep,  horse, 
and  ox  corpuscles,  while  the  B.  anthracis  is  non-hsemolytic.1 
The  former  form  no  capsule  in  the  animal  nor  when 
cultivated  in  an  inactivated  serum,  anthrax  forms  a 
capsule  in  such  circumstances. 

Pathogenicity. — The  anthrax  bacillus  is  pathogenic  for 
man,  cattle,  sheep,  goats,  rabbits,  guinea-pigs,  and  mice. 
The  horse  and  the  pig  are  also  susceptible ;  but  adult 
white  rats  are  partially,2  and  dogs,  cats,  and  Algerian 
sheep  are  completely,  immune. 

Inoculated  anthrax  is  rarely  fatal  to  cattle  in  India 
(Holmes). 

Young  white  rats,  or  rats  fatigued  by  muscular  work, 
can  be  infected,  and  frogs  and  fish,  though  immune  under 
ordinary  conditions,  can  be  rendered  susceptible  by  raising 
the  temperature  of  their  environment.  Birds,  such  as 
fowls  and  pigeons,  are  also  almost  insusceptible,  but  may 
be  rendered  susceptible  by  lowering  their  temperature  ; 
smaller  birds,  such  as  sparrows,  are  more  susceptible. 
The  virulence  varies  considerably  and  may  be  artificially 
modified  in  many  ways  :  by  passing  through  a  series  of 
susceptible  animals  it  is  heightened,  by  growing  in  the 
body  of  an  insusceptible  animal  it  is  lowered,  and  the 
latter  result  is  also  obtained  by  cultivating  for  two  or 
three  weeks  at  a  temperature  of  42°  to  45°  C.,  or  by  the 
addition  of  certain  chemical  substances  to  the  culture 
medium — for  example,  O01  per  cent,  of  potassium  bi- 
chromate. These  methods  of  "  attenuation,"  as  it  is 
termed,  are  practically  applied  in  the  preparation  of  the 
anthrax  vaccine. 

1  Jarmai,  Centr.  f.  Bakt.,  Abt.  I  (Orig.),  Ixx,  1913,  p.  72 

2  Hall,  Ibid.  Ixvi,  1912,  p.  293. 


SYMPTOMS  OF  ANTHRAX  257 

Symptoms  of  the  disease  in  cattle  are  not  very  marked. 
A  beast  may  appear  a  little  out  of  sorts  and  the  next 
day  be  found  dead,  or  after  suffering  for  a  day  or  two 
with  general  malaise,  fever,  and  rigors,  and  with  a  san- 
guineous discharge  from  the  nostrils  and  bowel,  it  dies 
suddenly.  Post-mortem,  the  chief  feature  that  attracts 
attention  is  enlargement  of  the  spleen ;  the  organ  may 
be  two  or  three  times  larger  than  normal,  is  highly  con- 
gested, and  very  soft  and  friable.  Microscopically,  the 
bacillus  is  found  in  enormous  numbers  in  the  spleen, 
somewhat  less  numerously  in  the  blood,  and  still  less  so 
in  the  liver,  kidney,  and  other  organs. 

Swine  do  not  often  suffer  from  this  disease,  unless  fed 
with  the  offal  of  an  infected  animal,  in  which  case  the  chief 
clinical  sign  is  great  enlargement  about  the  throat ;  this 
is  almost  pathognomonic,  while  the  chains  of  bacilli  tend 
to  be  somewhat  longer  than  in  other  animals. 

Mice  inoculated  subcutaneously  usually  die  in  about 
twenty-four  hours,  and  enlargement  and  congestion  of 
the  spleen  are  very  noticeable.  An  infected  guinea-pig 
generally  dies  in  about  thirty-six  hours  and  usually  shows 
no  symptoms  until  the  last,  when  it  may  suffer  from 
rigors,  with  high  temperature,  convulsions,  and  staring 
coat.  Post-mortem,  the  muscular  tissue  is  found  to  be 
pale  and  cedematous,  the  spleen  is  enlarged  to  two  or  three 
times  its  normal  size  and  is  highly  congested  and  very 
soft,  and  minute  haemorrhages  may  occur  in  the  serous 
membranes.  Microscopically,  bacilli  are  found  throughout 
the  spleen,  and  are  often  so  numerous  that  in  a  stained 
preparation  there  appear  to  be  more  bacilli  than  tissue. 
Large  numbers  are  also  present  in  the  blood  and  lungs, 
fewer  in  the  liver  and  kidney  ;  in  the  latter  organ  they  are 
almost  confined  to  the  glomeruli  (Plate  IV.  6).  Imme- 
diately after  death,  however,  comparatively  few  bacilli  may 
be  met  with  in  the  blood,  the  heart,  and  great  vessels. 

17 


258  A  MANUAL  OF  BACTERIOLOGY 

The  spread  of  the  disease  in  nature  seems  to  result 
from  the  ingestion  of  spores  while  the  animals  are  feeding. 
Although  the  bacilli  without  spores  would  be  destroyed 
by  the  acid  gastric  juice,  this  is  not  the  case  with  the 
spores,  which  are  probably  generally  developed  from  the 
organisms  present  in  the  bloody  discharges  of  a  stricken 
animal,  and  are  distributed  by  wind  and  flood,  and  in  this 
way  may  infect  large  tracts  of  pasture.  Crows  and  foxes 
may  also  serve  to  spread  the  disease  by  feeding  on  infected 
material  and  disseminating  the  spores  by  the  excreta.1 
Pasteur  suggested  that  earthworms  might  bring  the  spores 
to  the  surface  in  their  casts  from  the  buried  carcases  of 
infected  animals,  but  some  experiments  by  Koch  negatived 
this.  The  non-sporing  bacilli  rapidly  degenerate  and  die 
in  a  buried  carcase. 

Man  seems  to  be  relatively  insusceptible  to  anthrax. 
The  disease  is  generally  met  with  among  butchers, 
veterinary  surgeons,  shepherds,  etc.,  and  among  those 
who  sort  wool  or  hair  or  work  with,  or  carry,  hides,  e.g. 
glove-makers,  tanners,  porters,  etc.  The  disease  occurs 
in  two  forms :  the  so-called  "  malignant  pustule,"  a 
cutaneous  infection,  not  unlike  an  angry  carbuncle, 
occurring  at  the  seat  of  inoculation,  on  exposed  parts  of  the 
body,  such  as  the  back  of  the  neck,  the  face,  wrists,  and 
hands  ;  and  "  wool-sorters'  disease,"  a  general  infection, 
severe  and  fortunately  rare,  through  the  lungs  or  stomach. 
Rag-sorters  are  likewise  sometimes  attacked  by  anthrax, 
but  there  is  also  a  distinct  "  rag-sorters'  disease  "  which 
is  stated  to  be  due  to  a  non-motile,  non-sporing,  non- 
liquefying,  capsulated  bacillus,  the  Proteus  capsulatus 
hominis  2  of  Bordoni  Uffreduzzi. 

1  MoUet,  Centr.  f.  Bakt.,  Abt.  I  (Orig.),  Ixx,  1913,  p.  19. 

2  Capsulated  bacilli  have  been  met  with  in  many  septic  processes. 
This    group    includes    Friedlander's    pneumo-bacillus,    P.    capsulatus 
hominis,  B.  mucosus  capsulatus  of  Fricke,  and  the  B.  coli  immobilis. 
They  are  met  with  in  conditions  associated  with  sepsis,  pus  production, 


INDUSTRIAL  ANTHRAX  259 

Under  the  Factories  and  Workshops  Act  1895  all 
cases  of  anthrax  contracted  in  connection  with  various 
industries  have  now  to  be  reported  to  the  Home  Office. 
In  1909,  56  cases,  in  1910,  51  cases  were  thus  reported, 
with  mortalities  of  21*5  and  17' 6  per  cent,  respectively. 
In  addition,  in  1910  there  were  31  other  cases  in  England 
and  Wales.  101  cases  of  Anthrax  occurred  in  1913  with 
10  deaths  as  follows  : 

Industries  Cases  Deaths 

Wool 43  4 

Horsehair        ....         5  1 

Hides  and  Skins      .          .          .19  2 

Other  Industries       ...          3 
Not^  reportable          ...       31  3 

101  10 

Industrial  anthrax  has  also  been  exhaustively  dealt 
with  by  Legge.1  It  is  particularly  Persian  wool,  Chinese 
hides,  and  Russian  hair  which  are  dangerous,  while 
Argentine,  Australian,  and  New  Zealand  wools  are 
almost  innocuous.  The  sorting  and  exclusion  of  wool 
derived  from  infected  animals  seem  to  be  impracticable, 
and  the  efficient  sterilisation  of  the  thousands  of  bales 
that  are  imported  an  impossibility.  As  regards  hides 
and  skins,  Legge  points  out  that  it  is  doubtful  if  there  is 
any  way  in  which  hides  to  be  afterwards  tanned  can  be 
effectively  disinfected,  and  to  be  of  real  benefit  it  would 
have  to  be  done  before  the  material  is  opened  in  the 
warehouse  ;  but  to  secure  this  would  be  impossible.  A 
method  introduced  by  Seymour  Jones  has  been  favourably 
reported  on  2 ;  it  consists  in  soaking  the  skins  for  twenty- 
broncho-pneumonia,  ulcerating  stomatitis,  etc.  They  are  shortish,  non- 
motile,  non-sporing  rods,  usually  Gram-negative,  easily  cultivated  and 
not  liquefying  gelatin,  and  in  the  tissues  surrounded  with  a  capsule. 

1  Brit.  Med.  Journ.,  1905,  vol.  i,  pp.  529,  589,  and  641. 

2  Ponder,  Report  to  the  Worshipful  Company  of  Leathersellers,  1911. 


260  A  MANUAL  OF  BACTERIOLOGY 

four  hours  in  a  mixture  consisting  of  1  per  cent,  formic 
acid  and  1  in  5000  mercuric  chloride.  After  this  treatment 
the  skins  are  soaked  in  a  strong  brine  solution.  The 
writer,  however,  has  found  that  for  horse-hair  the  solution, 
to  be  efficient,  must  be  two  or  three  times  stronger 
than  this.  As  regards  horse-hair,  Webb  and  Duncan1 
carried  out  a  number  of  experiments  on  its  disinfection, 
from  which  it  would  seem  that,  leaving  out  of  considera- 
tion white  or  grey  hair,  which  is  liable  to  change  colour, 
no  injurious  effect  is  produced  on  hair  by  steam  disinfection 
provided  the  temperature  does  not  exceed  218°  F.  ;  but 
this  is  a  comparatively  low  temperature  for  efficient 
disinfection,  and  success  can  then  be  obtained  only  with 
minute  care  in  the  construction  and  regulation  of  the 
apparatus.  Legge  concludes  that  to  secure  certain  de- 
struction of  all  anthrax  spores  in  horsehair  absolute 
reliance  cannot  be  placed  on  either  steam  disinfection 
(within  the  limits  in  which  it  can  be  applied)  or  simple 
boiling.  Adoption  of  one  or  the  other  is  a  very  material 
safeguard,  but  risk  must  always  be  run  by  those  who 
prepare  the  hair  for  disinfection.  Disinfection  has  been 
attempted  by  subjecting  the  material  to  the  action  of 
certain  phenoloid  disinfectants,  but  from  experiments  by 
Hall  and  the  writer,  a  modified  Seymour-Jones  method 
or  formalin  or  bacterol  seem  to  be  the  only  efficient  ones.2 

Steam  disinfection  at  215°-230°  F.  can  be  applied  to 
wool,  but  the  fibres  are  materially  damaged  by  the  process.3 

A  number  of  cases  of  anthrax,  resulting  in  many  deaths, 
have  been  reported  in  various  parts  of  the  United  States 
from  tanneries  dealing  with  hides  imported  from  China. 

1  Ann.  Rep.  of  Chief  Inspector  of  Factories,  1900,  p.  472,  and  1902, 
p.  278. 

2  In  disinfection  experiments  with  anthrax,   agar  should  be  used 
for  the  subcultures,  broth  for  some  unexplained  reason  being  inefficient 
See  Hewlett  and  Hall,  Journ.  of  Hygiene,  xi,  1911,  p.  473. 

3  See  Eighth  Rep.,  Anthrax  Investigation  Board. 


ANTI-ANTHRAX  SERUM  261 

Also  a  number  of  cattle  have  been  infected  as  the  result 
of  drinking  water  from  rivers  and  creeks  receiving  the 
waste  liquors  from  these  works. 

Houston1  detected  the  anthrax  bacillus  in  a  catch-pit 
in  a  hide  factory  at  Yeovil,  and  in  sewage  and  effluents 
and  in  the  mud  of  the  Yeo.  It  has  also  been  met  with 
in  linseed  cake  and  oats. 

Toxins. — From  pure  cultures  of  the  Bacillus  anthracis 
Hoffa  obtained  small  quantities  of  a  ptomine,  which  pro- 
duced fall  of  temperature  and  haemorrhages,  and  Hankin 
isolated  a  proteose  which  in  large  amounts  was  fatal,  but 
in  small  amounts  conferred  immunity  to  subsequent 
inoculation  with  living  bacilli.  Brieger  and  Frankel 
obtained  a  tox-albumin  from  animals  dead  of  anthrax. 
Marmier,  by  growing  the  anthrax  bacillus  in  a  solution  of 
peptone,  glycerin,  and  salts,  and  subsequently  precipitating 
with  ammonium  sulphate,  obtained  a  toxin  which  he  states 
is  neither  protein  nor  basic,  and  is  contained  within  the 
bacterial  cells. 

Sidney  Martin,2  by  growing  the  anthrax  bacillus  in 
alkali  albumen  for  ten  days,  obtained  from  the  culture 
albumoses  and  an  alkaloidal  substance.  From  the  bodies 
of  animals  which  had  died  of  the  disease,  chiefly  from  the 
spleen  and  blood,  he  obtained  similar  substances,  the 
amount  of  alkaloid  being  more  than  double  that  of  albu- 
mose.  The  mixed  products  produced  fever  in  animals 
followed  by  coma  and  death.  The  albumose  was  proved 
to  be  the  fever,  and  the  alkaloid  the  coma,  producer ; 
the  latter  also  caused  a  spreading  oedema  at  the  seat  of 
inoculation. 

Anti-serum. — An  anti-serum  for  anthrax  was  prepared 
by  Marchoux  by  immunising  sheep  by  vaccination  and 
then  inoculating  with  progressively  increasing  doses  of 

1  Second  Rep.  Commis.  on  Sewage  Disposal,  1902,  p.  31. 

2  Brit.  Med.  Journ.,  1892,  vol.  i,  p.  641. 


262  A  MANUAL  OF  BACTERIOLOGY 

virulent  anthrax  cultures.  Sclavo  has  prepared  an  anti- 
serum  by  first  immunising  asses  with  a  vaccine  and  then 
inoculating  them  with  increasing  doses  of  virulent  cultures 
over  a  prolonged  period.  This  serum  has  been  used 
successfully  in  a  number  of  cases  of  anthrax  in  man,  and 
should  always  be  employed,  60-80  c.c.  being  injected 
intravenously.  Salvarsan  also  seems  to  be  an  efficient 
drug  for  the  treatment  of  anthrax.  As  already  mentioned 
(p.  239)  B.  pyocyaneus,  and  pyocyanase  obtained  there- 
from, is  antagonistic  to  anthrax  infection.  Louis  and 
Fortineau l  state  that  they  have  treated  50  cases  of 
anthrax  infection  in  man  by  injections  of  10  c.c.-20  c.c. 
of  sterilised  broth  cultures  of  B.  pyocyaneus  with  a 
mortality  of  10  per  cent. 

Vaccine. — An  attenuated  virus  has  been  extensively 
employed  for  the  prophylactic  vaccination  of  cattle  and 
sheep.  Cultures  are  attenuated  by  growing  at  42°-43°  C. 
(Pasteur,  Chamberland,  and  Roux).  A  weak  vaccine  is 
first  injected,  followed  after  ten  to  twelve  days  by  an 
injection  of  a  stronger  vaccine.  The  mortality  as  a  result 
of  the  vaccination  is  small  and  the  animals  are  subsequently 
protected  for  some  months  against  the  virulent  disease. 
Sobernheim  has  applied  a  combined  method,  5-15  c.c.  of 
anti-anthrax  serum  being  inoculated  on  one  side  of  the 
animal,  and  the  vaccine  on  the  other.  This  practically 
eliminates  all  danger  from  the  vaccine. 


Clinical  Examination 

(1)  In  veterinary  practice. — If  an  animal  is  suspected  to  have 
died  from  splenic  fever,  an  extensive  post-mortem  is  inadvisable 
because  of  the  risk  of  distribution  of  material  containing  bacilli 
with  subsequent  development  and  dissemination  of  spores,  with 
infection  of  pasture,  etc.  The  abdomen  should  be  opened  and  the 

1  Comp.  Rend.  Acad.  Sc.,  vol.  158,  No.  14,  1914,  p.  1035. 


MALIGNANT  PUSTULE  263 

spleen  examined.  If  this  is  found  to  be  much  enlarged,  and  so 
soft  that  it  can  hardly  be  handled  without  rupture,  there  is  a  high 
probability  of  splenic  fever,  which  the  history  of  sudden  death, 
with  or  without  symptoms,  coupled  with  a  sanguineous  discharge, 
increases.  To  confirm  the  diagnosis,  some  smear  preparations 
should  be  made  from  the  spleen  and  blood,  and  can  be  stained  and 
examined  on  arriving  home.  If  slides  or  cover-glasses  are  not 
available,  the  ear  or  a  small  piece  of  the  spleen  may  be  removed 
and  taken  home,  where  the  specimen  may  be  examined.  When 
material  is  sent  from  a  distance  for  examination  the  ear  should  be 
forwarded. 

The  smears  may  be  stained  with  Loftier 's  blue  and  by  Gram's 
method  with  eosin.  Methylene-blue  staining  gives  the  most 
characteristic  appearances,  according  to  McFadyean.  A  smear 
preparation  is  made,  not  too  thin,  is  air-dried,  and  then  fixed  by 
passing  once  through  the  Bunsen  flame.  The  film  is  stained  in  a 
1  per  cent,  aqueous  solution  of  methylene-blue  for  ten  minutes 
and  then  lightly  rinsed  and  dried.  The  anthrax  bacilli  appear  as 
blue  rods  surrounded  by  a  pale  violet  capsule.  If  the  post-mortem 
has  been  made  shortly  after  death  no  spores  are  visible.  Unless  the 
material  be  quite  fresh  large  saprophytic  bacteria  somewhat  resembling 
anthrax  are  always  present  and  must  not  be  mistaken  for  that 
organism  ;  by  the  McFadyean  method  of  stain  these  saprophytes 
do  not  show  the  violet  capsule.  If  a  hanging-drop  preparation  can 
be  made,  a  characteristic  is  the  non-motility  of  the  bacilli. 

The  stained  preparations  can  be  kept  and  produced  in  a  court  of 
law  if  necessary.  Cultivations  can  also  be  made  from  the  spleen, 
but  the  necessary  culture  media  are  not  of  course  usually  forth- 
coming. Finally,  a  guinea-pig  or  mouse  may  be  inoculated  sub- 
cutaneously  in  the  abdomen  with  a  particle  of  the  spleen,  and  after 
death  examined  microscopically  and  by  culture  methods. 

As  regards  the  disposal  of  the  carcase  of  an  animal  dead  from 
anthrax,  this  should  be  burned  if  possible,  but,  failing  this,  it  may 
be  buried  in  a  deep  pit,  preferably  with  plenty  of  lime.  All  traces 
of  blood  and  discharge  must  be  carefully  mopped  up  with  a  strong 
lime-wash  or  solution  of  chloride  of  lime,  or  other  reliable 
disinfectant. 

(2)  In  man. — In  malignant  pustule,  smear  specimens  should  be 
prepared  from  the  fluid  of  the  vesicles  or  with  the  scrapings  from  the 
incised  pustule,  or  sections  of  the  excised  pustule  may  be  made, 
and  stained,  some  with  Loffler's  blue,  others  by  Gram's  method 
with  eosin.  The  bacilli  are  not  often  met  with  in  the  blood,  except 
shortly  after  death.  Examination  of  the  blood-serum  of  the  case 


264  A  MANUAL  OF  BACTERIOLOGY 

by  the  opsonic  method,  using  anthrax  spores,  may  be  of  value. 
At  the  same  time  cultivations  on  agar  and  gelatin  should  be  pre- 
pared, and  may  yield  positive  results  when  the  microscopical 
examination  has  been  negative.  In  the  later  stages  of  the  disease 
the  bacilli  may  be  difficult  to  find,  even  in  sections. 

In  all  cases  of  doubt  a  guinea-pig  or  mouse  should  be  inoculated 
subcutaneously  with  the  material,  and  if  the  animal  dies  the  diagnosis 
of  anthrax  may  be  confirmed  by  the  characteristic  appearances,  by 
a  microscopical  examination,  and  by  cultivation.  The  animal 
experiment  is  by  far  the  most  certain  method  of  diagnosis,  a  nega- 
tive result  being  nearly  as  valuable  as  a  positive  one. 

N.B.  It  must  be  noted  that  both  cultivation  and  inoculation 
experiments  may  fail  to  give  positive  results  if  the  material  be  old 
or  putrid. 

(3)  In -wool,  hair,  etc. — Eurich  (loc.  cit.)  recommends  a  suitable 
quantity  of  the  material  to  be  placed  in  a  flask  with  50  c.c.  to 
100  c.c.  of  boiled  water  to  which  3-5  c.c.  of  5  per  cent,  solution  of 
caustic  potash  is  added.  If  much  blood-stained,  the  mixture  is 
allowed  to  stand  at  37°  C.  for  several  hours.  It  is  then  poured  into 
a  flat  dish  and  the  wool  or  hair  is  well  teased.  The  mixture  is  then 
heated  to  80°  C.  for  2-3  minutes.  Tubes  of  melted  agar  (6-9  c.c.) 
at  80°  C.  are  then  inoculated  with  ^  c.c.  of  the  wash  and  poured  into 
Petri  dishes  (4  inch).  The  characteristic  deep-lying  colonies 
(p.  253)  should  then  be  searched  for  after  twenty  hours'  incubation. 
Animals  may  also  be  inoculated. 


CHAPTER  VIII 
DIPHTHERIA  J 

Diphtheria  in  England — The  Diphtheria  Bacillus — The  Pseudo- 
Diphtheria  Bacillus — Clinical  Diagnosis — The  Xerosis  Bacillus 
— Diphtheritic  Affections  of  Birds  and  Animals 

DIPHTHERIA  seems  to  have  been  known  from  the  earliest 
ages,  being  recognised  by  the  classical  (medical)  writers, 
and  it  was  epidemic  in  England  and  on  the  Continent 
during  the  Middle  Ages.  Bretonneau2  experienced  an 
outbreak  at  Tours,  1818-1821,  and  gave  to  the  disease 
the  name  "  Diphterite  "  (afterwards  changed  to  "  Diph- 
terie ")  from  the  formation  of  membranes  which  is  so 
marked  a  feature  in  it.  In  England  the  diphtheria  deaths 
have  only  been  separately  scheduled  since  1855.  Since 
1881  until  recently  there  has  been  a  steady  increase  in  the 
prevalence  of  diphtheria,  particularly  in  the  large  towns, 
but  latterly  the  prevalence  seems  to  be  decreasing. 

As  regards  croup,  it  is  universally  admitted  that  the 
vast  majority  of  cases  of  membranous  croup  are  cases  of 
diphtheria. 

Diphtheria  is  distinctly  a  disease  of  the  young,  especially 
at  the  ages  from  two  to  ten,  and  this  holds  good  both  for 
London  and  for  England  and  Wales. 

That  diphtheria  is  an  infective  disease  is  amply  proved 
by  the  history  of  epidemics,  and  by  the  recorded  cases 

1  See  The  Bacteriology  of  Diphtheria,  Cambridge  University  Press, 
1908. 

2  See  Memoirs  on  Diphtheria,  New  Sydenham  Soc.,  1859. 

265 


266  A  MANUAL  OF  BACTERIOLOGY 

where  the  disease  has  been  conveyed  from  one  individual 
to  another. 

The  disease  occurs  in  all  grades  of  severity,  from  the 
classical  ones  with  wash-leather-like  membrane  and  great 
prostration,  to  those  which  present  a  mild  tonsillitis  or 
angina. 

The  bacteriological  study  of  diphtheria  was  commenced 
as  long  ago  as  1882  by  two  German  investigators,  Klebs 
and  Loffler.  Klebs  especially  investigated  the  pathological 
histology,  and  ascribed  the  disease  to  small  rod-shaped 
organisms,  which  he  observed  in  the  membrane.  It  was 
reserved  for  Loffler  to  place  this  observation  of  Klebs  on 
a  firmer  basis  by  the  isolation  and  cultivation  of  the 
bacillus  from  the  membrane,  and  by  the  production  of 
certain  phases  of  the  disease  by  inoculation  with  the 
isolated  organism.  The  cause  of  diphtheria  is,  therefore, 
this  diphtheria  bacillus,  which,  from  its  discoverers,  is 
frequently  known  as  the  Klebs-Loffler  bacillus. 

The  isolation  of  the  specific  organism  was  by  no  means 
an  easy  matter,  as  a  number  of  other  species  of  bacteria 
is  frequently  associated  with  it  in  the  membrane,  but  was 
accomplished  by  Loffler  by  the  use  of  a  special  culture 
medium  now  known  as  Loffler's  blood-serum,  which 
consists  of  a  mixture  of  blood-serum  (ox  serum  was  that 
originally  used)  3  parts  and  glucose  bouillon  1  part,  the 
whole  being  coagulated  (see  p.  61).  On  this  medium  the 
diphtheria  bacillus  grows  and  multiplies  exceedingly  well, 
while  the  other  organisms  associated  with  it  in  the  mem- 
brane are  to  a  large  extent  inhibited  in  their  growth.  By 
rubbing  a  small  piece  of  membrane  from  a  case  of 
diphtheria  over  the  surface  of  two  or  three  tubes,  or 
of  a  plate  of  Loffler's  serum,  and  incubating  at  37°  C. 
for  twenty  to  twenty-four  hours,  colonies  of  the  diph- 
theria bacillus  will  be  found  more  or  less  isolated  according 
to  the  number  of  organisms  present  in  the  membrane, 


DIPHTHERIA  267 

and  by  subculturing  from  these  pure  cultures  may  be 
obtained. 


Characters  of  the  Diphtheria  Bacillus 

Morphology. — The  B.  diphtheria  is  a  small,  delicate 
bacillus,  with  rounded  ends,  measuring  3  JUL  or  4  //.  in 
length.  It  is  non-motile  and  does  not  form  spores. 
The  size  varies  somewhat  even  on  the  same  medium, 
and  three  varieties  of  the  bacillus  have  been  described, 
viz.  long,  medium,  and  short,  according  to  the  length. 
These  varieties  tend  to  be  constant  and  to  breed  true. 
Some  of  the  rods  both  in  cultures  and  in  the  mem- 
brane have  a  swollen  end,  the  so-called  clubbing, 
and  parallel  grouping,  both  in  the  membrane  and  in 
cultures,  is  almost  universal,  the  bacilli  lying  parallel 
side  by  side  (Plate  VI.  a).  This  parallel  arrangement 
arises  from  the  peculiar  mode  of  division  of  the  bacillus. 
If  a  cell  be  observed  upon  a  warm  stage  it  first  elongates, 
then  becomes  constricted  at  about  its  middle,  and  then 
suddenly  one  side  of  the  cell-membrane  seems  to  rupture 
and  one  half  of  the  cell  bends  over  to  the  other,  so  that 
the  two  halves  form  a  V .  This  mode  of  division,  occurring 
in  contiguous  cells  and  being  repeated,  and  the  cells  thus 
becoming  more  and  more  crowded  together,  leads  to  the 
arrangement  in  parallel  series.  The  bacilli  are  generally 
joined  end  to  end  in  pairs,  and  distinct  thread  and  branch- 
ing forms,  though  of  rare  occurrence,  may  be  met  with. 
On  different  media  the  same  strain  exhibits  considerable 
variation  in  size.  On  blood-serum  and  on  gelatin  the 
bacilli  are  of  medium  length  and  on  the  whole  fairly  regular 
in  shape  ;  in  broth  they  tend  to  be  short  and  stunted  ; 
while  on  agar,  especially  glycerin  agar,  they  are  much 
larger  than  on  the  former  media,  and  long  club-shaped, 
spindle-shaped  and  barred  or  segmented  involution  forms 


268  A  MANUAL  OF  BACTERIOLOGY 

are  abundant ;  on  blood-serum  club-shaped  involution 
forms  also  occur,  but  sparsely  in  a  young,  eighteen  to 
twenty  hours'  culture,  in  a  forty- eight  hours'  culture 
more  numerously. 

Staining  reactions. — The  B.  diphtheria  stains  well  with 
the  ordinary  anilin  dyes  and  is  Gram-positive.  With 
Loffler's  methylene  blue  the  coloration  is  usually  some- 
what irregular,  more  deeply  stained  portions  alternating 
with  paler  intervals,  the  so-called  segmentation,  and 
especially  marked  with  agar  cultures.  The  ends  of  the 
organisms  are  also  frequently  deeply  stained,  the  so-called 
polar  staining,  while  the  phenomenon  known  as  "  meta- 
chromatism  "  is  often  marked  both  at  the  poles  and  also 
in  the  rod,  appearing  as  granules  of  a  purplish  tint  and 
contrasting  with  the  blue  of  the  methylene  blue.  With 
Neisser's  stain  (p.  294)  deep  inky  coloured  dots,  appearing 
somewhat  larger  in  diameter  than  the  rods,  occur  at  the 
poles  of  the  organism  and  occasionally  at  the  centre. 

Cultural  reactions. — The  diphtheria  bacillus  is  an  aerobic 
and  also  a  facultatively  anaerobic  organism,  and  grows 
well  on  all  the  ordinary  culture  media,  forming  cream- 
coloured  growths  or  colonies,  the  latter  on  serum  tending 
to  be  somewhat  flattened,  with  regular  margins.  It  grows 
slowly  on  gelatin,  forming  a  raised  whitish  growth  without 
liquefaction  of  the  medium,  and  flourishes  in  milk,  with 
the  production  of  an  acid  reaction,  but  without  curdling. 
In  broth  some  strains  give  a  granular  growth  on  the  sides 
and  at  the  bottom  of  the  tube,  the  broth  remaining  clear, 
sometimes  with  a  thin  surface  pellicle  ;  other  strains  may 
render  the  broth  turbid  throughout.  On  potato  the 
growth  is  slight  and  invisible. 

The  indole  reaction  can  be  obtained  in  peptone-water 
cultures  either  with  or  without  a  nitrite,  but  the  writer 
has  shown  that  this  reaction  is  due,  not  to  indole,  but  to 
skatolecarboxylic  acid  (see  below,  p.  288). 


PLATE  VI. 


a.  The   Klebs-Loffler  or  diphtheria   bacillus.     Film  preparation 
of  a  serum  culture,      x  1500. 


*  w   - 


•-**,".>' ":-N 

h^PviS. 


6.  Section  of  diphtheritic  membrane  with  Klebs-Loffler  bacilli.     Gram 
and  eosin.      x  750. 


DIPHTHERIA  269 

The  diphtheria  bacillus  attacks  glucose  and  lactose 
with  the  formation  of  acid  only,  no  gas  (see  Table,  p. 
292).  As  regards  the  production  of  acid,  Neisser  found 
that  during  the  first  nine  hours  there  is  little  or  none  ; 
at  the  end  of  twenty- four  hours  a  considerable  quantity 
has  been  formed,  and  the  amount  increases  until  the  end 
of  the  second  day,  after  which  the  production  ceases. 

The  B.  diphtheria  is  agglutinated  by  the  serum  of 
patients  and  by  a  diphtheria  serum,  but  the  test  is  difficult 
to  apply  on  account  of  the  coherence  of  the  growth,  is 
somewhat  erratic  with  different  strains,  and  is  of  no 
practical  value  in  the  diagnosis  of  the  disease.  For  the 
same  reasons,  the  agglutination  reaction  is  of  little  use 
for  the  recognition  of  the  organism  and  for  distinguishing 
it  from  the  so-called  "  pseudo-diphtheria  "  bacilli. 

The  Klebs-Loffler  bacillus  retains  its  vitality  in  cultures 
for  a  month,  and  when  dried  for  three  or  four  weeks. 
According  to  Welch  and  Abbot,  it  is  destroyed  in  ten 
minutes  by  a  temperature  of  58°  C.  It  is  readily  destroyed 
by  antiseptics  when  in  culture,  but  in  the  membrane  it  is 
difficult  to  find  an  agent  which  will  penetrate  and  kill 
the  bacilli  beneath  the  surface. 

The  diphtheria  bacillus  and  its  characters  under  culti- 
vation have  been  described  somewhat  fully,  because  of 
the  importance  of  the  identification  of  the  organism  as  a 
means  of  clinical  diagnosis.  As  mentioned  at  the  com- 
mencement of  this  chapter,  the  clinical  diagnosis  of  diph- 
theria presents  many  difficulties,  and  considerable  assist- 
ance may  be  derived  from  a  bacteriological  examination. 
The  diagnosis  is  based  on  the  presence  or  absence  of  the 
Klebs-Loffler  bacillus,  either  in  smears,  or  in  cultivations, 
made  from  the  membrane  or  secretion  (see  p.  292).  This 
method  is  of  very  real  assistance  in  doubtful,  and  especially 
in  mild,  cases,  which  clinically  it  may  be  very  difficult  to 
decide  whether  they  be  diphtheritic  or  no.  The  mild 


270  A  MANUAL  OF  BACTERIOLOGY 

cases  are  those  which  it  is  of  the  greatest  importance  to 
identify,  especially  in  schools,  for  if  not  recognised  the 
patients  may  go  about  and  prove  a  source  of  infection 
to  all  around.  The  method  also  affords  valuable  evidence 
as  to  when  a  case  can  be  considered  free  from  infection ; 
so  long  as  bacilli  are  present  in  the  throat  infection  must 
be  possible,  and  the  length  of  time  for  which  they  may 
occasionally  persist  is  remarkable.  In  half  the  cases  the 
bacilli  disappear  within  three  days  of  the  disappearance 
of  the  membrane,  in  a  few  cases  they  linger  for  as  long 
as  three  weeks,  but  occasionally  they  persist  much  longer. 
The  writer  isolated  them  for  so  long  as  five  months  (and 
virulent  to  the  last)  ;  and  a  case  is  recorded  in  which  they 
persisted  for  no  less  than  fifteen  months  after  the  attack. 
In  all  cases  two  or  three  examinations  should  be  made  at 
short  intervals  with  negative  results  before  the  bacilli 
can  be  pronounced  to  be  absent,  and  no  case  should  be 
discharged  from  hospital  until  the  absence  of  bacilli  has 
thus  been  proved.  When  bacilli  persist,  treatment  with 
antiseptic  sprays  or  gargles,  combined  with  syringing  the 
nose,  may  be  tried.  Syringing  the  nose  is  important,  for 
the  bacilli  probably  extend  to  the  post-nasal  space,  where 
they  are  untouched  by  a  throat  spray  or  gargle.  Another 
mode  of  treatment  has  also  been  adopted.  A  polyvalent 
anti-microbic  agglutinating  anti-diphtheria  serum  has  been 
prepared,  dried,  and  compressed  into  tablets,  one  of  which 
is  dissolved  in  the  mouth  every  two  hours,  and  fifteen 
minutes  after  solution  the  naso-pharynx  is  flushed  with 
physiological  salt  solution.  While  this  treatment  some- 
times succeeds,  it  often  fails.  The  writer  has  tried  the 
use  of  subcutaneous  inoculations  of  diphtheria  endotoxin 
(2-0-5-0  mgrm.)  at  intervals  of  seven  to  ten  days.  About 
half  the  cases  seem  to  clear  after  one  to  three  injections. 

With  regard  to  the  value  to  be  attached  to  the  bacterio- 
logical examination  for  diphtheria,  while  the  finding  of 


DIPHTHERIA  271 

the  bacilli  is  proof  positive  of  the  diphtheritic  nature  of 
the  affection  and  its  infective  nature,  their  apparent  absence 
is  not  of  so  much  value,  as  various  circumstances  modify 
the  result.  For  example,  an  unskilled  person  may  not 
happen  to  touch  the  right  spot  with  the  swab,  or  from 
struggling,  etc.,  on  the  part  of  the  patient  even  a  skilled 
operator  may  fail  to  reach  any  but  a  small  portion  of  the 
mucous  membrane,  instead  of  obtaining  a  good  mop  from 
all  over,  especially  when  there  are  no  definite  patches  of 
membrane.  The  use  of  antiseptic  gargles  or  paints  shortly 
before  the  swabbing  is  taken  will  likewise  prevent  the 
growth  of  the  bacilli.  It  sometimes  happens  that  a  very 
mixed  growth  is  obtained  in  the  cultures,  and  in  such 
cases  the  Klebs-Lofner  bacillus  may  be  missed.  Bearing 
such  sources  of  fallacy  in  mind,  and  making  due  allowances 
for  them,  the  negative  result  of  a  bacteriological  examina- 
tion may  have  considerable  value  in  those  cases  which 
clinically  are  doubtful.  In  no  case  where  there  is  a  reason- 
able suspicion  of  diphtheria  should  treatment  with  antitoxin 
be  delayed  until  the  bacteriological  report  is  obtained. 

The  bacilli  from  the  throat  are  frequently  associated 
with  other  organisms,  especially  micrococci  and  torulae  ; 
and  those  cases  in  which  the  temperature  tends  to  be  high 
and  the  throat  fetid  are  usually  a  mixed  infection  of 
diphtheria  bacilli  with  the  Streptococcus  pyogenes  or  Micro- 
coccus  pyogenes,  var.  aureus.  The  fact  of  such  mixed 
infection  cannot,  however,  be  definitely  decided  from  the 
cultures,  as  these  organisms  may  be  present  in  the  mouth 
or  throat  without  necessarily  taking  part  in  the  infective 
process.  Nor  can  the  severity  of  the  disease  be  gauged 
from  the  characters  or  numbers  of  the  diphtheria  bacilli 
and  other  organisms  present,  though  perhaps  in  a  number 
of  cases  those  which  yield  practically  pure  cultures  will 
probably  be  more  severe  than  the  cases  which  yield  cultures 
with  few  bacilli.  It  has  been  stated  that  the  long  form 


272  A  MANUAL  OF  BACTERIOLOGY 

of  the  diphtheria  bacillus  is  the  most,  and  the  short  form 
the  least,  virulent,  the  medium  being  intermediate,  but 
this  is  by  no  means  a  universal  rule.  Westbrook1  has 
divided  all  forms  of  the  diphtheria  bacillus  into  three 
groups,  distinguished  by  their  staining  reactions  with 
methylene  blue.  Those  with  deeply  staining  granules  he 
calls  "  granular  forms"  those  with  transverse  bands 
"  barred  forms"  and  those  staining  evenly  "  solid  forms" 
Each  group  is  further  divided  into  seven  types  according 
to  shape  and  size,  the  types  being  designated  by  the  letters 
A  to  G  and  being  progressively  smaller  from  A  to  G. 

It  is  sometimes  stated  that  a  microscopical  examination, 
unless  controlled  by  inoculation  of  the  isolated  bacteria,  is 
unreliable.  Such  a  statement  is  extremely  misleading. 
If  the  bacilli  which  have  been  cultivated  from  a  suspicious 
throat  possess  all  the  characters  of  diphtheria  bacilli, 
inoculation  experiments  are  not  needed,  and  if  they  were 
performed  with  a  negative  result  (i.e.  the  bacteria  are  not 
virulent)  would  prove  little,  for  the  bacilli  from  different 
parts  of  a  culture  from  a  throat  often  possess  different 
degrees  of  virulence.  Occasionally,  it  is  true,  even  the 
expert  may  be  in  doubt  about  a  particular  bacillus,  but 
such  cases  are  the  exception.  Here  an  inoculation  experi- 
ment may  help,  but  would  be  of  no  value  if  a  negative 
result  were  obtained.  It  is  absolutely  essential  in  the 
microscopical  examination  for  diphtheria  to  use  a  good 
lens,  proper  illumination,  and  sufficient  amplification,  not 
less  than  800-1000  diameters. 

Paihogenicity . — The  diphtheria  bacillus  is  pathogenic 
for  man,  the  horse,  ox,  rabbit,  guinea-pig,  cat,  chicken, 
pigeon,  and  finches,  all  of  which  are  more  or  less  susceptible, 
while  mice  and  rats  are  immune.  In  man  the  respiratory 
tract  is  usually  affected,  though  the  conjunctiva  and  other 
mucous  membranes,  as  of  the  vagina  and  stomach,  and 

1  Rep.  Minnesota  State  Board  of  Health,  1899-1900. 


DIPHTHEKIA  273 

wounds  may  be  attacked.  A  pseudo-membrane  usually 
forms,  consisting  of  laminae  of  fibrin  entangling  a  few 
leucocytes  and  other  cells,  and  here  and  there  small  effusions 
of  blood,  together  with  coagulative  necrosis  of  the  under- 
lying mucous  membrane,  and  the  bacilli  are  for  the  most 
part  located  in  the  superficial  layers  of  this  pseudo-mem- 
brane (Plate  VI.  6),  though  in  all  cases  in  which  the  disease 
has  lasted  for  any  time  they  are  found  in  the  lungs,  spleen, 
and  kidneys,  and  may  occur  even  in  the  blood.  If  the 
patient  recovers  from  the  diphtheritic  attack,  paralytic 
sequelae  are  not  uncommon  and  are  due  to  a  peripheral 
neuritis.  Pseudo-membranes  may  be  formed  by  other 
organisms,  e.g.  by  the  streptococcus  and  pneumococcus 
also  by  the  pneumobacillus,  and  occur  in  Vincent's  angina 
(p.  296),  but  it  is  doubtful  whether  paralytic  sequelae 
follow  any  but  a  diphtheritic  infection. 

Some  remarkable  skin  affections  of  an  eczematous  or 
ichthymatous  nature  have  been  found  by  Hare1  and 
others  to  be  due  to  the  diphtheria  bacillus. 

Another  affection  which  seems  to  be  generally  diphtheritic 
is  membranous  rhinitis.  Whereas  true  nasal  diphtheria  is 
a  serious  condition,  membranous  rhinitis  is  seldom,  if 
ever,  attended  with  any  risk  to  life,  sequelae  do  not  occur, 
and  it  is  rare  to  obtain  a  history  of  infection  from  cases 
of  it.  This  is  extraordinary  and  very  difficult  to  explain, 
for  virulent  diphtheria  bacilli  are  abundant  in  the  nose 
and  nasal  secretion. 

Diphtheroid  organisms  can  occasionally  be  isolated  from 
well  people  and  those  not  known  to  have  been  in  contact 
with  diphtheria  cases.  The  Klebs-Loffler  bacillus  can  be 
isolated  from  the  throats  of  nearly  7  per  cent,  of  the 
presumably  healthy  population  ;  2  in  the  throats  of  con- 
tacts the  percentage  rises  to  33  or  more.  Murray  and 

1  Lancet,  1908,  vol.  i,  p.  282. 

2  See  Eyre,  Brit.  Med.  Journ.,  1905,  vol.  ii,  p.  1104. 

18 


274  A  MANUAL  OF  BACTERIOLOGY 

the  writer  x  found  diphtheria-like  bacilli  in  58  out  of  385 
children  (15  per  cent.)  admitted  into  the  Victoria  Hospital, 
Chelsea. 

Ford  Robertson  believes  that  diphtheroid  organisms — 
possibly  the  Klebs-Loffler  bacillus  itself — may  play  an 
important  part  in  the  production  of  general  paralysis  of 
the  insane.  His  views  have  not  gained  general  acceptance, 
and  Eyre  (loc.  cit.)  found  that  the  percentage  incidence  of 
all  diphtheroid  organisms  and  of  the  Klebs-Loffler  bacillus 
in  the  throats  of  the  insane  was  not  greater  than  in  well 
persons,  and  was  unable  to  isolate  the  B.  diphtheria  post- 
mortem from  cases  of  general  paralysis. 

Traces  of  antitoxin  can  be  detected  in  the  blood  after 
an  attack  of  diphtheria,  usually  at  the  end  of  the  first  week 
of  convalescence :  this  antitoxin  has  probably  little  to  do 
with  the  actual  recovery  from  the  disease  (see  p.  208). 
A  small  amount  of  antitoxin  has  also  been  occasionally 
found  in  well  people  and  in  untreated  horses.  It  has 
been  suggested  that  in  such  cases  there  has  been  a  latent 
infection  with  the  B.  diphtheria,  but  on  Ehrlich's  side- 
chain  hypothesis  it  seems  more  likely  that  in  such  cases 
there  happens  to  be  an  excess  of  the  receptors  which 
constitute  antitoxin  naturally  free  in  the  blood. 

Guinea-pigs  are  the  animals  generally  employed  for 
experimental  work  on  diphtheroid  organisms.  In  order 
to  compare  the  effects  and  virulence  of  various  bacilli  it 
is  customary  to  make  the  inoculation  with  a  measured 
volume  of  a  forty-eight  hours'  broth  culture.  From  Ol 
c.c.  to  2  c.c.  of  such  a  culture,  according  to  the  virulence, 
inoculated  subcutaneously,  is  usually  required  to  kill  a 
250-grm.  guinea-pig  within  three  days.  At  the  seat  of 
inoculation  hsemorrhagic  oedema  forms,  haemorrhages 
occur  in  the  serous  membranes,  and  especially  in  the 

1  Brit.  Med.  Journ.,  1901,  vol.  i,  p.  1474.  See  also  Graham-Smith, 
Journ.  of  Hygiene,  vol.  iii,  1903,  p.  216. 


DIPHTHERIA  275 

adrenals,   while  the  renal  epithelium  and  the  liver-cells 
undergo  cloudy  degeneration. 

Inoculated  into  the  trachea  of  the  guinea-pig,  rabbit, 
and  chicken,  pseudo-membranes  form,  and  the  same  occurs 
with  the  superficially  injured  conjunctiva  and  vagina.  It 
is  stated  by  some  that  the  diphtheria  bacillus  does  not 
develop  on  a  normal  mucous  membrane — this  must  first 
be  injured,  and  the  staphylococcus  and  streptococcus,  so 
often  associated  with  the  diphtheria  bacillus  in  the  human 
subject,  may  play  a  part  in  preparing  the  way  for  infection 
by  damaging  the  cells  and  tissues.  Rabbits  usually  live 
somewhat  longer  than  the  guinea-pig  after  inoculation 
and  paralysis  frequently  develops  if  life  is  prolonged, 
simulating  the  post-diphtheritic  paralysis  of  man. 

The  question  of  the  occurrence  of  the  Klebs-Lofner 
bacillus  in  the  lower  animals  is  of  considerable  importance 
with  regard  to  the  spread  of  the  disease  and  the  conveyance 
of  infection.  The  so-called  diphtheritic  affections  of 
pigeons,  poultry,  and  calves  (referred  to  more  in  detail 
below,  p.  298)  are  as  a  rule  diseases  quite  distinct  from 
human  diphtheria,  and  are  not  communicable  to  man. 
A  number  of  observers  assert,  however,  that  cats  may 
suffer  from  the  disease,  which  in  these  animals  runs  a 
chronic  course,  and  is  associated  with  bronchitis,  lobular 
pneumonia,  nephritis,  and  wasting.  Klein 1  points  out 
that  not  only  are  cats  liable  to  the  disease  in  houses  where 
diphtheria  has  occurred,  but  that  a  similar  infectious  disease 
exists  naturally  among  cats,  and  symptoms  similar  to  this 
natural  disease  may  be  produced  by  inoculating  healthy 
cats  with  the  Klebs-Loffler  bacillus.  The  diphtheria  bacillus 
has  also  been  isolated  from  the  horse.2 

Several  epidemics  of  diphtheria  have  been  traced  to  an 
infected  milk  supply.  In  some  instances  the  infection 

1  Rep.  Med.  Officer  LOG.  Gov.  Board  for  1889,  p.  162. 

2  Cobbett,  Centr.  f.  Bakt.,  xxviii,  No.  19,  p.  631. 


276  A  MANUAL  OF  BACTERIOLOGY 

has  undoubtedly  been  derived  from  contamination  from 
a  human  source,  e.g.  in  an  outbreak  in  Lambeth,  Priestley 
traced  the  infection  to  the  ulcerated  thumb  of  an  employe 
in  a  particular  dairy  which  had  become  infected  with 
virulent  diphtheria  bacilli,  but  in  others  this  mode  of 
infection  has  not  been  demonstrated,  and  it  has  been 
suggested  that  certain  eruptive  conditions  on  the  teats 
and  udder  of  the  cow  may  be  caused  by  the  Klebs-Lofner 
bacillus  and  the  milk  become  infected  therefrom.  Klein l 
made  experiments  with  a  view  of  determining  this  point. 
He  inoculated  healthy  cows  in  the  shoulder  with  a  bouillon 
culture  of  the  diphtheria  bacillus.  This  caused  fever  and 
local  swelling,  and  in  about  a  week  a  papular  and  vesicular 
eruption  appeared  on  the  udders  and  teats.  The  B. 
diphtheria  was  isolated  from  the  contents  of  the  vesicles 
and  also  from  the  milk  on  the  fifth  day,  but  not  subse- 
quently. The  cows  died  in  two  to  four  weeks,  and  the 
B.  diphtheria  was  obtained  from  the  local  lesions.  Abbott 2 
obtained  somewhat  different  results,  but  Klein 3  points 
out  that  these  experiments  were  not  performed  under 
exactly  the  same  conditions  as  his  own. 

Klein,  Eyre,  Dean,  and  Marshall4  have  isolated  the 
diphtheria  bacillus  from  milk.  It  is  to  be  noted  that 
diphtheria-like,  but  non-pathogenic,  bacilli  are  often  to  be 
found  in  milk  and  cheese  (see  section  on  "  Milk  "). 

Toxins. — Diphtheria  toxin  has  not  been  obtained  in  a 
state  of  purity  and  its  exact  chemical  nature  is  unknown. 
LofHer  first  investigated  the  chemical  products  formed  by 
the  diphtheria  bacillus,  and  by  precipitating  bouillon 
cultures  with  alcohol  obtained  a  white  toxic  substance 
which  he  classed  among  the  enzymes. 

Roux  and  Yersin  precipitated  the  toxin  from  filtered 

1  Hep.  Med.  Officer  Loc.  Gov.  Board  for  1889  and  1890. 

2  Journ.  Path,  and  Bact.,  vol.  ii,  1894,  p.  35. 

3  Ibid.  p.  428. 

*  Jmirn.  of  Hygiene,  vol.  vii.  1907,  p.  32  (Refs.). 


DIPHTHERIA  277 

broth  cultures  by  means  of  absolute  alcohol,  and  also  by 
the  addition  of  calcium  chloride.  They  found  that  O4 
mgrm.  was  sufficient  to  kill  eight  guinea-pigs  or  two  rabbits, 
and  considered  it  to  be  an  enzyme. 

From  the  blood  and  spleen  of  cases  of  diphtheria  Sydney 
Martin 1  isolated  albumoses  (chiefly  deutero-albumose)  and 
an  organic  acid,  but  no  basic  body.  Injected  subcuta- 
neously  the  albumose  produces  much  oedema  and  irregu- 
larity of  temperature  ;  in  larger  doses  depression  of  tem- 
perature with  paralysis  and  coma.  Small  multiple  doses, 
not  sufficient  to  destroy  life,  may  give  rise  to  some  fever, 
and  in  two  or  three  days  to  paralysis  of  the  hind  legs  in 
rabbits,  with  general  weakness  and  loss  of  weight.  Post- 
mortem, the  nerves  are  found  to  have  undergone  degenera- 
tion— breaking  up  and  disappearance  of  the  myelin  and 
interruption  of  the  axis  cylinder,  while  the  heart  is  fatty. 
The  organic  acid  is  also  a  nerve  poison,  but  is  not  so  toxic 
as  the  albumose.  From  diphtheritic  membrane,  extracted 
with  a  10  per  cent,  salt  solution,  only  traces  of  albumose 
and  organic  acid  were  obtained,  but  the  extract  was 
highly  toxic,  producing  fever  and  paralysis.  Sidney 
Martin  suggests  that  a  substance  of  the  nature  of  a  ferment 
may  be  present,  and  that  the  ferment  in  the  membrane 
on  absorption  may  perhaps  form  the  albumose  in  the  body. 
From  cultures  of  the  diphtheria  bacillus  in  alkali-albumin, 
albumose  and  organic  acid,  with  similar  actions  to  those 
isolated  from  the  body,  were  obtained. 

Brieger  and  Frankel  (1890)  were  unable  to  find  any 
basic  substance  in  cultures,  and  concluded  that  the  toxic 
substance  was  a  protein  body,  which  they  designated  a 
"  tox-albumin."  It  was  destroyed  by  a  temperature  of 
60°  C.  but  not  by  one  of  50°  C.,  even  in  the  presence  of  an 
excess  of  hydrochloric  acid,  and  hence  is  probably  not  an 
enzyme.  The  tox-albumin  is  non-dialysable,  is  precipitated 

1  Brit.  Med.  Journ.,  1892,  vol.  i,  p.  641. 


278  A  MANUAL  OF  BACTERIOLOGY 

by  saturation  with  ammonium  sulphate  but  not  with 
magnesium  sulphate,  and  hence  is  neither  a  peptone  nor 
a  globulin,  contains  a  large  amount  of  sulphur,  and  gives 
the  biuret  and  Millon's  tests.  A  curious  property  of  this 
substance  is  that  small  quantities  (2'5  mgrm.  per  kilo- 
gramme of  the  body-weight)  do  not  produce  their  effects 
until  the  lapse  of  weeks.  Brieger  and  Boer  in  a  later 
research  prepared  the  diphtheria  tox-albumin  by  precipi- 
tating a  bouillon  culture  with  a  1  per  cent,  solution  of 
zinc  sulphate  or  chloride.  The  precipitate  of  the  zinc 
double  salt  was  washed  with  slightly  alkaline  water  and 
decomposed  with  a  stream  of  carbonic  acid  gas.  The 
purified  tox-albumin  gives  the  xanthoproteic,  biuret,  and 
Adamkiewicz's  reactions,  and  the  red  coloration  on  heating 
with  Millon's  reagent. 

According  to  Ehrlich  the  toxin  broth  is  a  complex 
mixture  of  toxic  constituents  belonging  to  the  proteins, 
but  this  is  denied  by  Madsen  and  Arrhenius  (see  p.  165). 
Its  poisonous  property  gradually  diminishes  on  keeping, 
and  is  destroyed  by  boiling  in  five  minutes,  at  lower 
temperatures  more  slowly,  and  also  by  light. 

Diphtheria  antitoxin. — By  the  injection  of  sub-lethal 
and  increasing  doses  of  the  toxin  into  an  animal  an  anti- 
toxin is  generated.  For  the  preparation  of  a  potent 
antitoxin  for  therapeutic  use  the  first  essential  is  a  highly 
toxic  toxin,  and  for  obtaining  this  a  diphtheria  bacillus  of 
high  virulence  is  required,  and  few  strains  possess  the 
necessary  virulence.  The  virulent  bacillus  is  grown  in  an 
alkaline  broth  (rendered  alkaline  to  the  extent  of  about 
5*7  c.c.  of  normal  caustic  soda  solution  per  litre  beyond 
the  neutral  point  of  litmus)  in  Erlenmeyer  flasks  containing 
half  to  one  litre  for  eight  to  twelve  days  at  37°  C.  Various 
small  details  have  to  be  attended  to  in  order  to  obtain  toxin 
of  maximum  toxicity  ;  it  is  important  that  growth  should 
occur  upon  the  surface  of  the  broth.  The  use  of  meat  some 


DIPHTHERIA  ANTITOXIN  279 

days  old  has  been  advocated,  or  of  acid  beef-broth  in  which 
B.  coli  has  been  grown  for  twenty-four  hours,  in  order 
to  eliminate  the  glucose  (p.  27).  L.  Martin  makes  use  of 
"  peptone "  prepared  by  the  auto-digestion  of  a  pig's 
stomach  with  dilute  hydrochloric  acid.  The  cultures  are 
then  filtered  through  a  Berkefeld  or  Pasteur-Chamberland 
filter  to  remove  the  bacilli.  The  filtrate  is  germ-free  and 
very  toxic,  and  a  little  carbolic  acid  may  be  added  to 
preserve  it.  In  New  York  10  per  cent,  of  a  5  per  cent, 
solution  of  carbolic  acid  is  added  to  the  culture,  the  bacilli 
are  allowed  to  deposit  by  standing  for  forty-eight  hours, 
and  the  culture  is  filtered  through  paper ;  in  this  way 
filtration  through  a  filter-candle  is  dispensed  with.  Less 
than  O'Ol  c.c.  of  the  toxin  should  kill  a  250-grm.  guinea- 
pig  in  three  to  four  days.  Selected  horses  which  have  been 
tested  with  mallein  and  tuberculin,  and  kept  under  obser- 
vation for  some  time  to  ensure  that  they  are  healthy,  are 
then  inoculated  with  this  filtrate,  commencing  with  a  dose 
of  O01  to  Ol  c.c.,  according  to  the  toxicity  of  the  toxin, 
or  20  c.c.  of  the  toxin  together  with  10,000  units  of  anti- 
toxin may  be  given  for  the  first  three  doses.  Individual 
horses  vary  very  much  in  their  susceptibility  to  the  toxin, 
so  that  care  has  to  be  exercised  with  the  first  injections. 
The  injections  are  given  subcutaneously  over  the  shoulder, 
and  produce  a  local  swelling  and  some  rise  of  temperature 
and  general  disturbance,  lasting  two  or  three  days.  When 
this  has  passed  away  the  inoculation  is  repeated,  a  larger 
dose  being  administered  provided  the  reaction  due  to  the 
former  one  was  not  too  severe.  The  treatment  is  con- 
tinued for  five  to  six  months,  the  dose  of  toxin  administered 
being  gradually  increased  until  it  may  attain  500  c.c.  or 
more.  Cartwright-Wood  found  that  by  growing  virulent 
diphtheria  bacilli  for  three  or  four  weeks  in  ordinary 
peptone  broth,  with  the  addition  of  10  or  20  per  cent,  of 
blood- serum  or  plasma,  subjecting  the  culture  to  a  tern- 


280  A  MANUAL  OF  BACTERIOLOGY 

perature  of  65°  C.  for  an  hour  and  filtering  before  injection, 
much,  larger  initial  doses  can  be  given  and  some  degree  of 
immunisation  attained,  and  subsequently  the  ordinary 
broth  cultures  may  be  injected  in  large  doses.  Individual 
horses  vary  much  in  their  capacity  to  yield  antitoxin  :  on 
the  whole  those  that  are  moderately  sensitive  to  the 
toxin  seem  to  produce  most  antitoxin ;  a  horse  to  be  of 
value  should  after  three  months'  treatment  yield  an  anti- 
toxic serum  containing  not  less  than  300  units  per  c.c. 
The  required  potency  having  been  attained,  as  shown  by 
the  test  described  below,  the  horse  is  bled  with  aseptic 
precautions,  the  blood  is  allowed  to  coagulate,  and  the 
serum  is  drawn  off  and  filled  into  sterile  bottles  each 
containing  a  dose  of  the  antitoxic  serum.  A  small  amount 
of  antiseptic,  such  as  trikresol,  is  generally  added  as  a 
precautionary  measure  to  prevent  the  multiplication  of  any 
stray  germs  that  may  have  gained  access  during  the 
various  manipulations. 

Standardisation  of  antitoxin. — The  potency  of  diphtheria 
antitoxin  is  always  described  in  "  units  "  and  is  estimated 
by  ascertaining  the  quantity  of  antitoxin  required  just 
to  neutralise  a  certain  amount  of  a  standardised  toxin 
when  both  are  injected  into  a  250-grm.  guinea-pig.  For- 
merly, by  Roux's  method,  the  minimal  lethal  dose  of  the 
toxin  is  first  ascertained,  and  then  the  number  of  grammes 
of  guinea-pig  which  1  c.c.  of  antitoxin  will  protect  against 
this  minimal  lethal  dose  is  determined.  If  0*01  c.c.  of 
antitoxin  protects  a  300-grm.  guinea-pig  against  the 
minimal  lethal  dose,  1  c.c.  will  protect  300  x  100  =  30,000 
grm.  of  guinea-pig,  and  the  immunising  value  of  the  anti- 
toxin would  be  described  as  30,000.  This  method  is  open 
to  the  fallacy  that  if  only  a  portion  of  the  lethal  dose  be 
neutralised  the  guinea-pig  may  survive,  and  a  fictitious 
value  be  given  for  the  potency  of  the  antitoxin.  Behring 
later  adopted  ten  minimal  lethal  doses  as  the  test  dose 


STANDARDISATION  OF  ANTITOXIN        281 

of  toxin,  and  he  termed  ten  times  the  amount  of  antitoxin 
which  protects  a  guinea-pig  against  the  ten  minimal  lethal 
doses  a  unit  (the  Behring  unit,  which  therefore  =  100 
minimal  lethal  doses  of  toxin),  from  which  the  Ehrlich 
unit,  now  universally  adopted,  is  derived.  Though  this 
method  eliminates  to  a  large  extent  the  objections  to  the 
Roux  method,  Ehrlich  found  that  by  it  the  same  antitoxin 
tested  with  different  toxin  broths  yielded  different  values. 
This  he  explained  by  assuming  that  diphtheria  toxin  broth 
contains  not  only  toxin  but  also  other  substances  which 
combine  with  antitoxin.  These  substances,  though  non- 
toxic,  or  comparatively  so,  vary  in  amount  in  different 
toxin  broths,  and  variable  results,  therefore,  may  be 
obtained  by  the  simple  method  of  testing.  These  sub- 
stances, having  an  affinity  for  antitoxin,  are  toxoids 
and  toxone.  There  are  several  varieties  of  toxoids,  viz. 
(1)  those  having  a  greater  affinity  for  antitoxin  than  toxin 
itself,  protoxoids ;  (2)  those  having  the  same  affinity, 
syntoxoids  ;  (3)  and  those  having  a  less  affinity,  epitoxoids.1 
Toxoids  are  probably  derivatives  of  toxin  ;  they  increase 
in  quantity  in  old  toxin  broth  which  has  been  kept,  and 
which  at  the  same  time  decreases  in  toxicity.  The  toxones 
also  combine  with  antitoxin,  having  a  less  affinity  for  it 
than  toxin,  are  primary  secretory  products  of  the  diphtheria 
bacillus,  and  while  not  acutely  lethal,  induce  induration, 
necrosis,  and  paralysis.  The  toxoids  are  comparatively 
scanty  in  a  fresh  toxin  broth  and  are  negligible,  but  it  is 
otherwise  with  the  toxone,  which  is  always  present  in 
appreciable  quantity.  Owing  to  the  fact  that  toxone  has 
less  affinity  for  antitoxin  than  toxin  has,  if  an  exactly 
neutral  mixture  of  toxin  broth  and  antitoxin  be  prepared, 
considerably  more  than  the  minimal  lethal  dose  of  the  toxin 
broth  must  be  added  to  render  the  mixture  acutely  toxic, 

1  See  pp.  165-168  for  other  views  on  the  constitution  of  diphtheria 
toxin. 


282  A  MANUAL  OF  BACTERIOLOGY 

because  the  first  portion  of  the  added  toxin  simply  dis- 
places the  toxone  from  its  combination  with  the  antitoxin, 
and  is  neutralised  by  the  antitoxin  so  set  free. 

Thus,  suppose  a  certain  amount  of  a  toxin  broth  contains 
90  units  of  toxin  and  10  units  of  toxone,  and  to  this  amount 
100  units  of  antitoxin  are  added  so  as  to  form  a  physiolo- 
gically neutral  mixture,  the  combination  which  occurs  is 
shown  by  the  following  "  equation  "  :  90  toxin-antitoxin  -f- 
10  toxone-antitoxin  =  L0  (i.e.  neutrality).  If  an  amount 
of  the  toxin  broth  be  now  added,  corresponding  to  11  units 
of  toxin,  the  effect  will  be  as  though  only  one  unit  of  toxin 
has  been  added,  as  is  shown  by  the  following  "  equation  "  : 
90  toxin-antitoxin  +  10  toxone-antitoxin  +  11  toxin  = 
100  toxin-antitoxin  +  10  toxone  (free)  +  1  toxin  (free)  = 
L+  (i.e.  just  acutely  lethal).  Thus  although  the  equivalent 
of  eleven  minimal  lethal  doses  of  toxin  has  been  added  to 
the  physiologically  neutral  mixture  of  toxin  broth  and 
antitoxin,  only  one  minimal  lethal  dose  of  toxin  remains 
free  and  active,  because  ten  toxin  units  displace  the  ten 
toxone  units  from  the  toxone-antitoxin  complex  and  are 
neutralised  by  the  antitoxin  thus  set  free.  Ehrlich,  there- 
fore, devised  a  method  of  standardisation  which  eliminates 
irregularities  due  to  the  variable  proportions  of  toxone 
and  toxin  in  the  toxin  broth  by  adopting  antitoxin  and 
not  toxin  as  the  standard.  In  order  to  standardise  an 
antitoxin,  a  virulent  toxin  broth  is  employed  and  its 
minimal  lethal  dose  is  approximately  ascertained — i.e. 
that  amount  which  is  just  sufficient  to  kill  a  250-grm. 
guinea-pig  on  the  fourth  or  fifth  day.  A  solution  of  accu- 
rately standardised  antitoxin,  which  can  be  obtained  from 
the  Serumspriifung  Institut,  Frankfort-on-Maine,  is  then 
prepared,  containing  one  "  unit  "  of  the  antitoxin  in  1  c.c., 
and  the  toxin  is  standardised  with  this  by  mixing  with 
one  unit  various  quantities  above  and  below  one  hundred 
minimal  lethal  doses.  It  is  required  to  ascertain  the 


STANDARDISATION  OF  ANTITOXIN          283 

amount  of  the  toxin  broth  which,  when  mixed  with  one 
unit  of  antitoxin,  just  suffices  to  kill  a  250-grm.  guinea- 
pig  on  the  fourth  or  fifth  day  after  the  injection  of  the 
mixture ;  this  amount  of  toxin  is  known  as  the  L+  dose. 
The  L+  dose  may  be  defined  as  that  amount  of  a  given 
diphtheria  toxin  broth  which  is  not  completely  neutralised 
by  one  "  unit "  of  standard  antitoxin  to  the  extent  that 
exactly  one  simple  lethal  dose  of  toxin  remains  unneutralised ; 
it  corresponds  usually  to  105-120  minimal  lethal  doses. 
For  example,  suppose  0'003  c.c.  of  the  toxin  was  found 
to  be  the  minimal  lethal  dose,  with  separate  "  units  "  of 
standard  antitoxin,  0*2,  0'3,  0*4,  and  0'5  c.c.  respectively 
of  the  toxin  might  be  mixed,  and  each  mixture  injected 
into  a  guinea-pig  ;  probably  the  guinea-pigs  receiving  the 
"  unit  "  of  antitoxin  plus  0'2  and  0'3  c.c.  of  toxin  would 
remain  alive,  while  the  animal  receiving  the  0'4  c.c.  of 
toxin  would  die  in  twenty-four  to  forty-eight  hours.  The 
death  in  the  last  case  is  too  rapid  ;  more  than  a  simple 
lethal  dose  has  remained  unneutralised,  and  therefore  the 
L+  dose  of  toxin  lies  between  0'3  and  0'4  c.c.,  and  further 
experiments  would  have  to  be  performed  with  amounts 
of  toxin  between  these  limits  in  order  to  ascertain  the 
exact  dose.  Death  of  the  guinea-pig  on  the  fourth  or 
fifth  day  has  been  chosen  because  it  has  been  found  that 
if  the  dose  of  toxin  be  diminished  ever  so  little  below  that 
producing  this  result,  death  does  not  ensue  under  nine  or 
ten  days.  That  is  to  say,  an  acute  intoxication  is  fatal 
at  the  latest  on  the  fourth  or  fifth  day,  a  fatal  result  after 
then  being  due  to  a  chronic  intoxication.  The  amount  of 
toxin  which  is  exactly  neutralised  by  one  "  unit "  of  the 
standard  antitoxin  is  known  as  the  L0  dose.  By  exact 
neutralisation  is  meant  absence  of  any  reaction,  general  01 
local,  at  the  seat  of  inoculation,  in  the  inoculated  guinea- 
pig.  If  toxin  broth  were  a  single  substance,  containing 
only  toxin,  then  L,  -  L0  =  D,  the  simple  lethal  dose, 


284  A  MANUAL  OF  BACTERIOLOGY 

would  be  equal  to  the  minimal  lethal  dose.  But  because 
of  the  presence  of  toxone,  which  also  has  an  affinity  for 
antitoxin,  D,  the  difference  between  the  L+  and  the  L0 
doses,  is  usually  a  multiple  (8-12)  of  the  minimal  lethal 
dose. 

From  these  considerations  we  are  now  in  a  position 
to  define  the  unit  of  antitoxin  :  a  "  unit  "  is  that  amount 
of  antitoxin  which  will  neutralise  about  100  minimal  lethal 
doses  for  the  guinea-pig  of  diphtheria  toxin.  From  certain 
considerations  Ehrlich  considers  that  the  unit  would 
exactly  neutralise  200  minimal  lethal  doses  of  a  theoretical 
toxin,  containing  only  toxin  and  neither  toxoid  nor  toxone, 
but,  inasmuch  as  such  a  toxin  is  unknown  practically,  the 
unit  corresponds  usually  to  105-120  minimal  lethal  doses 
of  a  toxin  broth,  the  extremes  which  Ehrlich  has  found 
being  16  and  136  lethal  doses.  Having  standardised  a 
specimen  of  toxin  by  means  of  standard  antitoxin,  this 
standardised  toxin  is  in  its  turn  used  to  standardise  the 
antitoxic  serum  which  has  been  prepared  for  therapeutic 
use.  The  toxin  is  preserved  by  the  addition  of  toluol, 
and  is  kept  in  a  cool,  dark  place ;  it  needs  to  be  restand- 
ardised  every  few  weeks. 

In  standardising  antitoxin,  the  L+  dose  of  the  stand- 
ardised toxin  is  mixed  with  varying  amounts  of  the 
antitoxin,  the  mixtures  are  injected  into  guinea-pigs,  and 
the  amount  of  the  antitoxic  serum  which  neutralises  the 
L+  dose  of  toxin  is  thus  ascertained.  If,  for  example,  it 
were  found  that  0'05,  OO4,  and  0'03  c.c.  of  the  antitoxic 
serum  neutralised  the  L+  dose  of  toxin,  but  that  the  guinea- 
pig  receiving  0'025  c.c.  suffered  from  some  local  necrosis, 
wasted,  and  died  in  a  few  days,  and  the  animal  receiving 
0*02  c.c.  died  in  two  or  three  days,  0'03  c.c.  of  this  anti- 
toxin would  be  about  equivalent  to  one  unit  of  standard 
antitoxin,  and  the  antitoxic  serum  therefore  contains  33 
units  per  c.c.  For  all  the  experiments  the  conditions 


ANTITOXIN  TREATMENT  285 

must  be  kept  as  constant  as  possible,  guinea-pigs  weighing 
250  grm.  or  thereabouts  employed,  and  to  eliminate  irregu- 
larities a  number  of  animals  must  be  used.  The  antitoxic 
constituent  of  diphtheria  antitoxin  is  globulin  in  nature, 
or  is  intimately  associated  with  the  globulin  content  of 
the  serum.  Thus  Atkinson  found  that  if  the  serum  is 
precipitated  by  saturation  with  magnesium  sulphate,  the 
whole  of  the  antitoxin  is  carried  down  with  the  precipitate, 
and  also  that  the  globulin  content  of  the  blood- serum  of 
antitoxin  horses  is  increased.  His  results  were  confirmed 
by  Ledingham.1 

There  can  now  be  no  doubt  as  to  the  value  of  the  antitoxin 
treatment  of  diphtheria.  Since  the  introduction  of  antitoxin 
treatment,  which  was  commenced  about  the  middle  of  1894,  there 
has  been  a  steady  decline  in  the  case  mortality  from  diphtheria, 
especially  in  London,  where  probably  the  majority  of  the  cases 
are  injected  with  antitoxin.  From  1891  to  1894  the  case  mortality 
from  diphtheria  in  the  hospitals  of  the  Metropolitan  Asylums  Board 
averaged  about  30  per  cent,  in  1895  it  was  22-8  per  cent.,  and  after- 
wards steadily  fell,  until  during  the  last  eight  years  it  has  ranged 
between  8-3  and  10  per  cent. 

Not  less  than  2000  units  should  be  injected  for  a  dose,  and  early 
treatment  is  of  paramount  importance.  As  soon  as  there  is  a 
reasonable  probability  that  the  case  is  one  of  diphtheria  the  anti- 
toxin should  be  used,  and  treatment  should  not  be  delayed  for  the 
result  of  the  bacteriological  examination.  The  statistics  show  that 
in  cases  treated  on  the  first  day  of  the  disease  the  case  mortality 
is  3-3,  on  the  second  day  it  is  6-5,  on  the  third  day  10-6,  on  the 
fourth  day  12-9,  and  on  the  fifth  day  and  afterwards  14-8  per  cent. 

In  bad  cases,  and  in  those  coming  under  treatment  at  a  late 
stage  of  the  disease,  the  dose  may  be  increased  to  10,000,  20,000, 
or  even  30,000  units  with  advantage,  and  to  bring  the  patient  under 
the  influence  of  the  antitoxin  as  rapidly  as  possible  the  first  dose 
may  be  administered  intravenously.  The  dose  may  have  to  be 
repeated  once  or  twice  in  mild  cases,  in  bad  cases  perhaps  every 
six  or  twelve  hours  until  several  doses  have  been  given,  the  guide 
being  the  general  condition  of  the  patient  and  the  rapidity  of  the 
separation  of  the  membrane.  In  addition  to  antitoxin,  the  recum- 

1  Journ.  of  Hygiene,  vol.  VA»  *   007,  p.  65. 


286  A  MANUAL  OF  BACTERIOLOGY 

bent  posture  and  general  and  local  treatment  should  be  pursued 
as  usual. 

In  cases  of  mixed  infection,  in  which  the  diphtheria  bacilli  are 
associated  with  streptococci  or  staphylococci,  diphtheria  antitoxin 
may  prove  of  less  value,  as  it  has  no  influence  on  the  streptococcic 
or  staphylococcic  infection,  and  injections  of  anti-streptococcic 
serum  may  be  given  in  addition. 

Diphtheritic  paralysis  seems  to  be  rather  more  frequent  after  the 
use  of  antitoxin  than  in  the  cases  not  treated  with  it,  probably 
because  a  greater  number  of  cases  survive. 

The  antitoxin  has  also  been  employed  as  a  prophylactic  in  schools 
or  other  places  where  susceptible  individuals  are  congregated  together, 
and  where  cases  of  diphtheria  have  occurred,  with  excellent  results. 

The  procedure  in  such  circumstances  should  consist  of  a  bacterio- 
logical examination  of  the  throats  of  all  the  inmates  in  the  institu- 
tion, isolation  of  those  in  whom  the  B.  diphtheria  is  found,  and  the 
injection  of  every  one,  or  at  least  of  all  the  young  contacts,  with  a 
prophylactic  dose,  repeated  if  considered  desirable,  ten  days  later. 
For  this  purpose  a  dose  of  about  500  units  should  be  given.  The 
immunity  so  produced  does  not  last  for  more  than  three  weeks. 

The  objection  to  the  use  of  antitoxin  for  prophylaxis  is  that 
should  the  patient  subsequently  develop  diphtheria,  treatment  with 
antitoxin  may  induce  serious  symptoms  due  to  supersensitisation  or 
anaphylaxis.  To  obviate  this,  an  antitoxin  prepared  in  the  ox  has 
been  placed  on  the  market  for  prophylactic  use.  The  writer 
believes  that  all  the  advantages  of  antitoxin  without  its  disadvan- 
tages may  be  obtained  by  the  use  of  a  vaccine  consisting  of  diph- 
theria endotoxin,  and  that  it  is  of  service  in  the  treatment  of  carrier 
cases. x  Behring  2  has  suggested  the  use  of  a  toxin-antitoxin  mix- 
ture for  prophylactic  use  and  the  treatment  of  carrier  cases.  This, 
although  non-toxic  for  the  guinea-pig,  engenders  the  formation  of 
a  large  amount  of  antitoxin  in  the  recipient  which  persists  for  a 
long  time. 

Some  clinicians  assert  that  antitoxin  exerts  its  effect  when 
administered  by  the  mouth  or  the  rectum.  Hewlett  was  unable  to 
detect  any  absorption  of  tetanus  antitoxin  from  the  stomach  or 
rectum,  nor  Sternberg  of  diphtheria  antitoxin  from  the  rectum,  of 
rabbits.  Blumenau  and  Dzerzhgovsky  could  in  no  instance  secure 
immunity  in  animals  by  oral  administration  of  diphtheria  antitoxin, 
nor  could  any  antitoxin  be  detected  in  the  blood  of  animals  so 
treated  (Roussky  Vratch,  March  9,  1913). 

1  Lancet,  July  20,  1912,  and  June  28, 1913. 

2  Deut.  Med.  Woch.,  May  8,  1913. 


PSEUDO-DIPHTHERIA  287 

Pseudo-diphtheria  and  Diphtheria-like  Bacilli 

Diphtheria-like  bacilli  are  not  uncommon  in  wounds 
and  in  pathological  exudates,  etc.,  and  in  connection  with 
diphtheria  an  important  question  must  be  discussed,  viz. 
the  occurrence  and  nature  of  the  so-called  pseudo-diphtheria 
bacilli.  The  term  was  originally  used  by  Loffler,  and  by 
the  rule  of  priority  should  be  reserved  for  the  organism 
described  by  him  under  this  name.  The  pseudo-diphtheria 
bacillus  of  all  authors  is  an  organism  occurring  in  the 
throat  in  various  anginal  conditions,  scarlet  fever,  etc., 
and  occasionally  in  the  throats  and  noses  of  well  persons, 
and  is  non-pathogenic  to  guinea-pigs.  Park  and  Beebe 
met  with  it  in  twenty-seven  out  of  330  healthy  throats 
examined  by  them.  Roux  and  Yersin,  Abbott  and  Frankel 
describe  it  as  morphologically  resembling  the  Klebs- 
Loffler  bacillus,  while  Loffler,  von  Hofmann,  Koplick, 
Park  and  Beebe,  Peters,  and  Hewlett  and  Miss  Knight,1 
consider  that  an  organism  differing  somewhat  from  the 
Klebs-Loffler  bacillus  should  alone  be  termed  the  pseudo- 
diphtheria  bacillus ;  to  avoid  confusion  it  is  best  to 
designate  it  the  Hofmann  bacillus. 

Morphology. — Typically,  the  Hofmann  bacillus  is  a 
shortish  rod  tapering  towards  the  ends,  which  are  rounded, 
the  average  length  being  from  1'5  ^  to  2  /*,  and  it  occurs 
in  pairs,  resembling  two  suppositories  placed  base  to  base. 
It  is  non-motile,  does  not  form  spores,  is  arranged  in  a 
parallel  grouping  like  the  Klebs-Loffler  bacillus  (due  to  the 
same  mode  of  division),  and  involution  forms  are,  as  a 
rule,  not  met  with  (Plate  VII.  a).  It  is  Gram-positive, 
and  stains  deeply  and  regularly  with  Loffler' s  methylene 
blue,  segmentation  and  polar  staining  usually  being  absent. 
With  Neisser's  stain  no  inky  granules  are  perceptible,  as  is 
the  case  with  the  diphtheria  bacillus. 

1  Trans.  Brit.  Inst.  of  Prev.  Med.,  vol.  i,  1897. 


288  A  MANUAL  OF  BACTERIOLOGY 

Cultural  reactions. — The  Hofmann  bacillus  develops 
well  at  temperatures  from  20°  to  37°  C.,  and  is  almost  a 
strict  ae'robe  ;  there  is  no  growth  anaerobically  in  hydrogen. 
On  serum,  agar,  and  gelatin  it  forms  cream-coloured  colonies 
or  growths,  barely  distinguishable  from  those  of  the  Klebs- 
Loffler  bacillus  ;  gelatin  is  not  liquefied.  On  ordinary 
potato  it  hardly  grows  at  all,  what  growth  there  is  being 
quite  invisible.  On  alkaline  potato,1  however,  it  forms 
distinct  cream-coloured  colonies,  usually  visible  by  the 
second  day.  Tn  stab-cultures  in  gelatin  and  glucose-agar 
no  gas  is  formed,  and  the  growth  is  confined  to  the  upper 
part  of  the  stab.  In  broth  it  forms  sometimes  a  granular 
deposit,  sometimes  a  general  turbidity.  On  neutral  litmus 
glucose-agar  and  in  litmus  milk  a  blue  colour  is  developed, 
indicating  the  production  of  alkalinity ;  milk  is  not 
curdled.  Cultivated  in  peptone  water  an  indole-like 
reaction  with  sulphuric  acid  alone  can  be  obtained  after 
a  variable  time,  three  to  four  weeks,  while  the  diphtheria 
bacillus  gives  it  in  about  a  week  ;  with  a  nitrite  and 
sulphuric  acid  the  indole-like  reaction  can  be  obtained  with 
both  the  pseudo-  and  diphtheria  bacilli  in  about  a  week. 
The  substance  giving  this  indole-like  reaction  is  not  indole, 
but  skatole-carboxylic  acid.2  A  broth  culture  reduces  a 
weak  solution  of  methylene  blue.  The  Hofmann  bacillus 
is  non-pathogenic  to  guinea-pigs  in  doses  of  5  c.c.  or  more 
of  a  forty- eight  hours'  broth  culture,  but  is  virulent  to 
certain  birds  (see  below,  p.  290).  Mandelbaum  and  Heine- 
mann  3  state  that  if  a  glycerin- agar  plate  be  smeared  with 
human  blood  and  inoculated,  the  diphtheria  bacillus 
produces  colonies  surrounded  by  a  yellow  zone,  while  the 
colonies  of  the  Hofmann  and  xerosis  bacilli  do  not  change 

1  Ordinary  potato  rendered  alkaline  with  a  10  per  cent,  solution  of 
sodium  carbonate  before  sterilisation. 

2  Hewlett,  Trans.  Path.  Soc.  Land.,  vol.  li,  1900,  p.  187  ;    vol.  lii, 
1901,  p.  113. 

3  Centr.  f.  Bakt.  (Orig.),  liii,  1910,  p.  356. 


PLATE  VII. 


a.  The   pseudo-diphtheria   or   Hofmann   bacillus.     Film 
preparation  of  a  serum  culture,      x  1500. 


b.  Vincent's  angina.     Smear  from  exudation  showing  fusiform 
bacilli  (dark)  and  spirilla  (light),      x  2000. 


THE  HOFMANN  BACILLUS  289 

the  red  colour  of  the  blood.  In  addition,  the  Hofmann 
bacillus  does  not  ferment  any  sugar,  etc.  (see  Table,  p.  292). 

The  histories  of  several  cases  investigated  by  Miss 
Knight  and  Hewlett  seemed  to  show  that  the  Hofmann 
bacillus  is  associated  with  mild  anginal  conditions,  which 
are  free  from  complications,  end  in  recovery,  and  are  not 
followed  by  sequelae.  In  many  of  the  cases  the  anginal 
condition  was  associated  with  distinct  patches  of  mem- 
brane, and  in  two  symptoms  were  present  suggestive  of 
the  toxaemia  which  is  met  with  in  diphtheria. 

In  a  long  series  of  experiments  Hewlett  and  Miss  Knight 
believed  that  some  evidence  was  obtained  of  the  conversion 
of  the  Hofmann  into  the  Klebs-Loffler  bacillus  and  vice 
versa.  Moreover,  the  Hofmann  bacillus  seemed  in  many 
instances  to  replace  the  Klebs-Loffler  bacillus  in  the  throat 
during  convalescence,  and  it  is  possible  in  a  large  series  of 
cultures  to  obtain  connecting  links  between  the  Klebs- 
Loffler  bacillus  on  the  one  hand  and  the  Hofmann  bacillus 
on  the  other.  Cobbett,1  however,  suggests  that  these  facts 
are  capable  of  another  explanation,  viz.  that  during  the 
acute  stage,  diphtheria  bacilli  being  readily  found,  the 
Hofmann  bacillus  is  likely  to  be  overlooked,  whereas  at  a 
later  stage  a  more  careful  search  may  be  necessary  to 
detect  the  diphtheria  bacillus,  and  in  the  course  of  that 
search  the  Hofmann  bacillus  is  therefore  more  frequently 
seen. 

Miss  Knight  and  Hewlett  came  to  the  conclusion  that 
in  some  cases,  at  least,  the  Hofmann  bacillus  is  a  modified 
Klebs-Loffler  bacillus,  and  the  view  taken  of  its  relation 
to  the  Klebs-Loffler  bacillus  was,  that  it  is  a  very  attenuated 
Klebs-Loffler  bacillus,  i.e.  one  far  removed  from  virulence. 
It  would  therefore  seem  wise  to  treat  anginal  cases  in  which 
the  pseudo-diphtheria  bacillus  is  found  as  possibly  infective, 
though  it  would  probably  be  inexpedient  to  admit  to  a 

1  Journ.  of  Hygiene,  vol.  i,  1901. 

19 


290  A  MANUAL  OF  BACTERIOLOGY 

general  diphtheria  ward  (unless  a  prophylactic  dose  of 
antitoxin  or  of  an  endotoxic  vaccine  be  given),  nor  would 
antitoxin  be  needed  in  the  majority. 

Most  authorities  have  been  unable  to  convert  the  pseudo- 
bacillus  into  a  virulent  Klebs-Loffler  bacillus,  or  vice  versa, 
and  many  are  of  opinion  that  it  has  probably  nothing  to 
do  with  diphtheria  (Park  and  Beebe,  Peters,  Washbourn, 
Cobbett,  Clark).  A  few  fatal  cases  have  been  recorded 
(e.g.  by  Stanley  Kent)  in  which  a  careful  search  has  failed 
to  reveal  any  but  Hofmann  bacilli.  Boycott 1  found  that 
the  seasonal  prevalence  of  the  Klebs-Loffler  and  Hofmann 
bacilli  does  not  correspond,  the  former  prevailing  during 
September,  October,  and  November  ;  the  latter  is  more 
frequent  from  May  to  August. 

Priestley  records  an  outbreak  of  what  he  terms  "  pseudo- 
diphtheria,"  in  which  the  Hofmann  bacillus  seemed  to 
be  the  causative  organism,  and  expresses  the  opinion 
that  this  bacillus  is  not  related  to  the  Klebs-Loffler 
bacillus.2 

Salter  3  claimed  to  have  found  that  the  Hofmann  bacillus 
is  virulent  to  many  small  birds  (goldfinch,  chaffinch,  canary, 
etc.),  and  that  by  successive  passages  it  becomes  converted 
morphologically  into  a  Klebs-Loffler  form  with  feeble 
virulence  for  the  guinea-pig.  He  also  found  the  filtered 
broth  culture  of  the  Hofmann  bacillus,  though  harmless  to 
guinea-pigs,  to  be  toxic  to  small  birds,  and  that  it  contains 
a  non-toxic  substance  (toxoid)  which  has  the  power  of 
combining  with,  and  neutralising,  diphtheria  antitoxin. 
Salter  concluded,  therefore,  that  diphtheritic  organisms 
are  to  be  met  with  of  every  grade  of  virulence,  the  weakest, 
known  as  Hofmann's  or  the  pseudo- diphtheria  bacillus, 
representing  the  most  attenuated  form  of  the  Klebs-Loffler 

1  Journ.  of  Hygiene,  1905,  vol.  v,  p.  223. 

2  Public  Health,  July  1903. 

3  Trans.  Jenner  Inst.  Prev.  Med.,  vol.  ii,  p.  113.     (Bibliog.) 


THE  HOFMANN  BACILLUS  291 

bacillus.  The  writer,1  Cobbett,2  Petrie,3  Williams,4  and 
Clark 5  have,  however,  quite  failed  to  confirm  Salter's 
results.  Thiele  and  Embleton  also  claim  to  have  effected 
the  transformation  of  a  typical  Hofmann  bacillus  into  a 
virulent  Klebs-Loffler  bacillus  by  massive  intra-peritoneal 
inoculation  of  guinea-pigs  with  Hofmann  culture  suspended 
in  30  per  cent,  gelatin  and  after  death  of  the  guinea-pig, 
injection  of  the  peritoneal  exudate  with  a  smaller  amount 
of  living  bacilli  into  a  second  guinea-pig,  and  repeating 
this  method  of  inoculation.  Finally  the  bacillus  became 
Klebs-Loffler  in  morphology  and  1  c.c.  of  its  toxin  killed  a 
guinea-pig  in  forty-eight  hours,  and  this  toxin  was 
neutralised  by  diphtheria  antitoxin. 

To  sum  up  :  the  Klebs-Loffler-like  avirulent  bacilli  met 
with  in  the  throat,  the  pseudo-diphtheria  bacilli  of  Roux 
and  Yersin,  are  probably  modified  and  avirulent  diphtheria 
bacilli.  As  regards  the  Hofmann  bacillus,  the  general 
trend  of  opinion  at  present  is  to  consider  it  as  quite  distinct 
from  the  Klebs-Loffler  bacillus.  Another  view  is  to  regard 
it  as  in  reality  including  several  species,  of  which  one 
may  be  a  modified  Klebs-Loffler  bacillus,  the  others  having 
no  relation  with  this  organism.  The  Klebs-Loffler-like 
avirulent  bacilli  might,  therefore,  be  regarded  as  true 
diphtheria  bacilli  slightly  removed  from  virulence,  the 
Hofmann  bacillus,  if  derived  from  the  Klebs-Loffler,  as  a 
diphtheria  bacillus  far  removed  from  virulence. 

In  determining  the  fermentation  reactions  of  the  diphtheria-like 
bacilli,  the  organisms  should  first  be  grown  in  broth  until  they 
become  acclimatised  to  this  medium,  or  should  be  grown  in  a 
medium  which  suits  them,  e.g.  broth  with  the  addition  of  serum  or 
of  ascitic  fluid.  Hiss's  serum- water  medium  is  satisfactory — serum 

1  Brit.  Med.  Journ.,  Sup.,  July  9,  1904. 

2  Journ.  of  State  Med.,  vol.  xi,  p.  609. 

3  Journ.  of  Hygiene,  vol.  v,  p.  134. 

*  Journ.  Med.  Research,  1902,  p.  83. 

5  Journ.  Infect.  Diseases,  vol.  vii,  1910,  p.  335. 


292 


A  MANUAL  OF  BACTERIOLOGY 


1  part,  water  3  parts,  with  1  per  cent,  of  carbohydrate  or  other 
substance,  tinged  with  litmus  and  sterilised  in  the  steamer  on  three 
consecutive  days.  Graham-Smith  *  gives  the  following  Table  of 
fermentation  tests  : 


Hiss's  medium  (10  days'  growth). 

Organism. 

C 

1 

§' 

I 

1 

_• 

O 

1 

i 

03 

1 

1 

| 

'5 

c 

1 

Q 

0> 

£ 

0 

B.    diphtherice,    virulent 

C 

C 

C 

C 

C 

C 

C 

and  avirulent    . 

A 

A 

A 

A 

A 

A 

A 

Hofmann  bacillus  * 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Xerosis  bacillus  * 

C 
A 

0 

0 

0 

0 

C 
A 

0 

0 

C 
A 

C 

C 

r 

B.  coryzce  * 

A 

0 

0 

A 

0 

\j 

A 

0 

0 

0 

Diphtheria-like    bacilli  : 

From  the  ear  * 

0 

0 

0 

0 

0 

0 

0 

0 

0 

From  the  urethra  * 

A 

0 

0 

A 

A 

A 

0 

0 

0 

From  the  throat  * 

C 
A 

0 

0 

C 
A 

A 

C 
A 

0 

0 

0 

From  the  fowl  * 

A 

0 

0 

A 

A 

0 

0 

0 

(*  Avirulent  to  the 

guinea-pig) 

C  =  coagulation  ;  —  =  no  coagulation  ;  A  =  acid  ;  0  =  no  reac- 
tion. Slight  variations  were  occasionally  noted  :  for  example,  four 
out  of  twenty  diphtheria  bacilli  gave  no  acid  with  lactose,  and  the 
amount  of  acid  production  and  of  coagulation  was  somewhat  variable. 


Clinical  Diagnosis 

(A)  In  man  and  animals  : — I.  In  some  cases  the  diphtheria 
bacillus  can  be  identified  in  the  membrane  or  discharge,  and  the 
diagnosis  established  thereby. 

Films  are  made  with  the  exudation,  or  with  a  fragment  of  the 
membrane  teased  up  as  finely  as  possible  on  a  slide,  a  droplet  of 
water  being  added  if  necessary.  One  of  these  films  should  be 

1  Journ.  of  Hygiene,  vol.  vi,  1906,  p.  286. 


DIAGNOSIS  OF  DIPHTHERIA  293 

stained  with  Loffler's  methylene  blue,  another  by  Gram's  method. 
The  bacilli  will  be  found  lying  parallel  to  one  another  in  larger  or 
smaller  groups,  together  with  involution  forms.  Films  stained 
with  Neisser's  or  Pugh's  stain  (see  below)  may  also  be  of  assist- 
ance. Another  method  is  to  stain  the  films  for  five  seconds  in 
dilute  carbol-methylene  blue  (seven  drops  to  10  c.c.  water), 
rinsing  and  drying,  and  counter-staining  in  dilute  carbon-fuschin 
(ten  drops  to  10  c.c.  water)  for  one  minute,  rinsing  and  drying 
(Higley). 

*  II.  Frequently  the  membrane  is  so  crowded  with  different  forms 
of  organisms  that  it  is  extremely  difficult  to  recognise  the  diphtheria 
bacilli  with  any  degree  of  certainty.  Recourse  must  then  be  had  to 
cultivation. 

For  this  purpose  sloping  blood-serum  tubes,  or  tubes  of  serum- 
agar,  must  be  employed  ;  simple  agar  is  unsuitable. * 

A  piece  of  membrane  or  a  swabbing  from  the  throat  is  rubbed 
over  the  surface  of  one  or  two  serum  tubes,  care  being  taken  not 
to  break  up  the  medium.  The  tubes  are  incubated  at  37°  C.  for 
eighteen  to  twenty  hours,  and  are  then  examined  microscopically 
whether  there  is  any  visible  growth  or  not.  If  there  be  no  visible 
growth  a  scraping  is  taken  by  means  of  a  sterilised  platinum  needle 
from  the  whole  surface  and  a  film  is  prepared.  If  there  is  a  visible 
growth  the  film  should  be  prepared  from  the  most  likely  colonies, 
or,  if  the  growth  be  confluent,  from  the  upper  half  inch  or  so. 
A  microscopical  examination  must  always  be  made,  for  some 
colonies — certain  staphylococci  and  torulae,  for  example — simulate 
those  of  the  diphtheria  bacillus  very  closely.  The  films  may  be 
stained  with  Loffler's  methylene  blue  for  five  to  ten  minutes,  or 
by  Pugh's  method,  then  washed  and  dried.  If  the  films  are  made 
on  a  slide,  after  staining,  washing,  and  drying,  a  drop  of  cedar  oil 
may  be  put  on  the  stained  patch,  which  is  then  examined  directly 
without  a  cover-glass.  If,  however,  there  is  very  little  growth,  it 
is  better  to  make  a  cover-glass  specimen,  as  the  position  of  the 
material  is  so  much  more  easily  located.  The  preparations  are 
examined  with  a  iVm-  oil-immersion  lens  magnifying  not  less  than 
800-1000  diameters,  and  the  Klebs-Loffler  bacillus  is  identified 
from  the  description  given  in  the  text. 

Prausnitz  considers  that  if  negative  results  are  obtained  after 
eighteen  to  twenty -four  hours'  incubation  the  tubes  should  be  incu- 
bated for  a  further  twenty  to  twenty -four  hours  and  re-examined, 

1  Various  selective  media  have  been  devised,  e.g.  potassium-sulpho- 
cyanide,  neutral-red,  glucose- blood-serum  (Rankin,  Journ.  of  Hyg. 
xii,  1912,  p.  60). 


294  A  MANUAL  OF  BACTERIOLOGY 

and  undoubtedly  occasionally  a  positive  result  may  be  obtained 
by  this  longer  incubation. 

Loffier's  methylene  blue  gives  much  more  characteristic  prepara- 
tions than  Gram's  method. 

Although  eighteen  to  twenty  hours  is  recommended  for  incubating 
the  cultures,  a  microscopical  examination  will  sometimes  reveal 
the  bacilli  at  a  much  earlier  period.  The  writer  has  found  them  in 
as  short  a  time  as  six  hours,  but  if  bacilli  are  then  not  found  the 
tubes  must  be  incubated  for  the  longer  period. 

Neisser's  method  of  staining  is  as  follows  : 

(a)  One  gramme  of  methylene  blue  (Griibler's)  is  dissolved  in 
20  c.c.  of  96  per  cent,  alcohol,  which  is  then  mixed  with  950  c.c. 
of  distilled  water  and  50  c.c.  of  glacial  acetic  acid. 

(b)  Two  grammes  of  Bismarck  brown  are  dissolved  in  one  litre 
of  boiling  distilled  water  and  the  solution  is  filtered. 

The  preparations  are  stained  in  (a)  for  one  to  three  seconds, 
rinsed  in  water,  and  stained  in  (b)  for  three  to  five  seconds,  washed 
in  water,  dried,  and  mounted.  The  bacilli  are  stained  brown,  and 
contain  two,  rarely  three,  inky-blue  dots.  This  is  a  valuable  con- 
firmatory stain  for  the  diphtheria  bacillus,  but  staining  for  a  longer 
time  than  that  recommended  by  Neisser  is  advisable,  viz.  half  a 
minute  in  the  blue  and  one  minute  in  the  brown.  Tanner  treats 
with  Gram's  iodine  solution  for  half  a  minute  after  the  blue.  The 
staining  solutions  seem  to  keep  well  but  occasionally  fail  to  act,  so 
should  be  controlled  on  an  undoubted  diphtheria  culture. 

Pugh's  stain  is  also  a  very  good  one.  It  is  a  mixture  containing 
1  grm.  of  toluidine  blue  dissolved  in  20  c.c.  of  absolute  alcohol 
and  added  to  1000  c.c.  of  distilled  water  and  20  c.c.  of  glacial 
acetic  acid.  The  mixture  is  applied  for  two  minutes.  The  proto- 
plasm of  the  bacilli  is  stained  a  pale  blue  and  the  polar  bodies  are 
deeply  stained  and  stand  out  in  marked  contrast  ;  by  artificial 
light  they  appear  a  reddish  purple. 

In  the  majority  of  cases,  after  a  little  experience,  the  Klebs- 
Loffler  bacillus  will  be  readily  recognised  if  present.  Occasionally, 
however,  bacilli  may  be  present  which  resemble  the  Klebs -Loftier 
very  closely,  and  of  which  it  is  difficult  to  be  certain.  In  such  a 
case  the  following  points  should  be  noted  in  attempting  to  arrive 
at  a  decision  : 

1.  The  character  of  the  growth  on  the  medium. 

2.  The  depth  of  staining  with  Loffler's  blue,  and  the  presence  or 
absence   of   segmentation    or    polar    staining :     the   Klebs-Loffler 
bacillus  usually  stains  somewhat  deeply,  while  the  bacilli  resembling 
it  stain  but  feebly. 


DIAGNOSIS  OF  DIPHTHERIA  295 

3.  The  presence  or  absence  of  involution  forms,  clubbing,  etc. 

4.  The  presence  or  absence  of  thread  forms  :    the  Klebs-Loffler 
bacillus  does  not  form  threads.1 

5.  The  presence  or  absence  of  spores  :    the  Klebs-Loffler  bacillus 
does  not  form  spores. 

6.  Motility  in  a  hanging  drop  :    the  Klebs-Loffler  bacillus  is  non- 
motile. 

7.  Gram's  method  of  staining  :    the  Klebs-Loffler  bacillus  stains 
well. 

8.  The  grouping  of  the  organism  :    the  parallel  grouping  of  the 
Klebs-Loffler  bacillus  is  somewhat  characteristic.     The  bacilli  when 
lying  side  by  side  do  not  seem  quite  to  touch,  while  the  bacilli 
which  resemble  the  Klebs-Loffler  and  show  a  parallel  grouping 
frequently  lie  much  closer  together  than  the  Klebs-Loffler  bacillus 
ever  does. 

9.  The  reaction  with  Neisser's  or  Pugh's  stain  (the  culture  must 
be  a  young  serum  one) :    the  pseudo-bacillus  and  other  bacilli  do 
not  give  the  diphtheritic  reaction  (polar  staining). 

10.  The  final  test  of  virulence  may  be  applied.     For  this  pur- 
pose the  organism  must  be  isolated  in  pure  culture  by  plate  cultiva- 
tions.    Two  guinea-pigs,  of  250-300  grm.  weight,  are  each  inocu- 
lated with  2  c.c.  of  a  forty-eight  hours'  broth  culture,  one  receiving 
at  the  same  time  1  c.c.  of  diphtheria  antitoxin.     If  the  guinea-pig 
inoculated  with  culture  only  dies,  while  the  one  receiving  culture 
and  antitoxin  lives,  this  is  complete  proof  that  the  organism  is  the 
diphtheria  bacillus  ;   if  both  live  no  inference  can  be  made  except 
that    the  organism  is  non-virulent ;    if    both    die   it  shows    that 
the    organism    is    virulent,     but    that    it    is    not    neutralised    by 
antitoxin,  and  therefore  is  not  the  diphtheria  bacillus.     In  cases  in 
which  bacilli  persist,  the  test  of  virulence  is  frequently  applied.     If 
the   organism  proves   to   be   non-virulent,  presumably  the  patient 
is  non-infective.     Such   a  presumption,    in    the    writer's    opinion, 
however,  is  not  necessarily  true. 

11.  Agglutination  tests  are  unsatisfactory  and  not  of  service. 

It  occasionlly  happens  that  a  conclusion  cannot  be  arrived  at 
without  an  extended  investigation. 

If  serum  tubes  are  not  available  an  egg  may  be  used.  It  is 
boiled  hard,  the  shell  chipped  away  from  one  end  with  a  knife 
sterilised  by  heating,  and  the  inoculation  made  on  the  exposed  white  ; 
the  egg  is  then  placed,  inoculated  end  down,  in  a  wine-glass  of  such 

1  Klein  and  others  have  described  thread  and  branched  forms  in 
cultures  of  the  Klebs-Loffler  bacillus  in  certain  circumstances,  but 
these  are  not  likely  to  be  observed  under  the  conditions  mentioned. 


296  A  MANUAL  OF  BACTERIOLOGY 

a  size  that  it  rests  on  the  rim  and  does  not  touch  the  bottom.  A  few 
drops  of  water  may  with  advantage  be  put  at  the  bottom  of  the 
glass  to  keep  the  egg-white  moist.  The  preparation  is  kept  in  a 
warm  place  for  twenty-four  to  forty-eight  hours  and  then  examined. 
Antitoxin  itself  may  be  used  as  a  culture  medium,  provided  it  con- 
tains no  antiseptic  (this  is  now  rarely  the  case).  A  test-tube  is 
sterilised  by  heating,  or  with  boiling  water  or  steam  from  a  kettle, 
antitoxin  to  the  depth  of  about  an  inch  is  poured  in,  and  is  coagulated 
by  holding  the  tube  very  obliquely  in  boiling  water  or  steam.  After 
coagulation  and  cooling  the  medium  is  inoculated.  If  no  incubator 
is  available,  the  culture  may  be  kept  in  a  warm  place,  or  in  an 
inside  pocket. 

Many  laboratories  now  undertake  the  examination  of  material. 
Culture  outfits  are  supplied  by  some,  consisting  of  a  sterilised  tube 
containing  a  sterilised  swab.  Failing  this,  a  piece  of  membrane 
may  be  forwarded  in  a  tube  or  bottle  which  has  been  sterilised  by 
heating,  or  with  boiling  water  or  steam.  If  there  be  no  membrane, 
a  swab  can  be  readily  extemporised  by  wrapping  a  little  wool  or 
lint  (non-antiseptic]  round  the  end  of  a  piece  of  wire,  knitting 
needle,  hair-pin,  penholder,  or  splinter  of  wood.  The  wood  may 
be  sterilised  by  moistening  with  water  and  then  holding  in  a  flame. 
Membrane  or  secretion  may  also  be  forwarded  on  pledgets  of  wool, 
pieces  of  lint  or  calico,  and  even  on  paper,  but  these  are  not  so 
suitable. 

(B)  In  milk.— See  section  on  "  Milk." 


Vincent's  Angina 

An  infective  malady  characterised  by  sore  throat,  fetor, 
dysphagia,  and  ulceration  and  membrane  simulating  diphtheria, 
The  diphtheria  bacillus,  however,  is  not  present,  and  the  affection 
is  caused  by  an  apparent  association  of  a  bacillus  and  a  spirochaete. 
The  bacillus  (B.  fusiformis)  measures  6-8  p.  to  10-12  p.  in  length, 
has  pointed  ends  and  is  usually  somewhat  bent,  not  straight,  often 
appears  feebly  motile,  and  does  not  stain  by  Gram.  It  can  be 
cultivated  anaerobically  on  the  ordinary  media  to  which  human 
blood-serum,  ascitic  or  hydrocele  fluid  has  been  added.  The 
spirochaete  is  long  and  sinuous  and  very  motile,  but  cannot  be 
cultivated,  and  is  stated  to  be  developed  from  the  fusiform  bacillus, 
Smears  may  be  stained  with  methylene  blue  or  dilute  carbol-fuchsin, 
and  the  appearance  of  the  associated  organisms  is  so  characteristic 
that  a  diagnosis  is  easily  effected  (Plate  VII.  b). 


THE  XEROSIS  BACILLUS  297 

Fusiform  bacilli  have  been  met  with  in  various  necrotic  pro- 
cesses, e.g.  noma  (see  Chapter  XX). 


The  Xerosis  Bacillus 

The  xerosis  bacillus  was  isolated  by  Neisser  from  cases  of  xerosis 
conjunctives,  and  is  met  with  in  follicular  conjunctivitis.  Lawson 
and  also  Griffith  isolated  it  from  nearly  50  per  cent,  of  all  normal 
conjunctival  sacs.  In  morphology  and  staining  reactions  it  re- 
sembles the  Klebs-Loffler  bacillus  very  closely.  It  differs  from  the 
Klebs-Loffier  bacillus  in  the  following  particulars  :  (1)  Usually, 
but  not  always,  in  the  primary  cultivations  from  the  eye  on  blood- 
serum,  colonies  do  not  appear  under  about  thirty  hours,  while 
those  of  the  Klebs-Loffler  bacillus  are  visible  in  sixteen  to  twenty 
hours.  This  does  not  apply  to  the  secondary  cultivations,  in  which 
the  colonies  appear  as  soon  as  those  of  the  Klebs-Loffler  bacillus. 

(2)  Upon  agar  it  will  seldom  or  never  grow  in  primary  culture,  and 
in  secondary  cultures  it  forms  only  a  thin,  translucent,  dry  film. 

(3)  Upon  gelatin  it  will  never  grow  in  primary  culture  and  seldom 
in  secondary  culture.     (4)  It  does  not  give  rise  to  acid  production 
in  milk  or  glucose  broth.     (5)  It  is  non-pathogenic  to  guinea-pigs. 
(6)  The  Neisser  stain  is  negative.     The  fermentation  reactions  will 
be  found  in  the  Table  on  p.  292. 

In  all  probability  the  organism  is  not  causative  of  xerosis  con- 
junctivse. 

To  isolate  the  organism,  blood-serum  tubes  are  inoculated  with 
a  looped  platinum  needle  from  cases  of  follicular  conjunctivitis  or 
xerosis  and  incubated  at  37°  C.  for  forty  to  forty-ei  ght  hours. 
Half  the  tubes  will  usually  show  a  growth.  Preparations  may  be 
stained  with  Loffler's  blue  and  by  Gram's  method. 


Bacillus  coryzae  (segmentosus) 

An  organism  first  described  by  Cautley,  of  frequent  occurrence 
in  the  nasal  secretion  in  cases  of  "  influenza  "  cold.  It  bears  a 
striking  resemblance  morphologically  to  the  B.  diphtheria  when 
stained  with  methylene  blue,  and  is  Gram -positive,  but  does  not 
show  granules  either  with  Loffler  blue  or  with  Neisser's  stain.  On 
agar  it  grows  more  slowly  than  B.  diphtherice,  and  in  glucose  broth 
and  litmus  milk  acid  production  is  slow  and  feeble.  It  is  non- 
pathogenic  to  guinea-pigs.  The  fermentation  reactions  will  be 
found  in  the  Table  on  p.  292. 


298  A  MANUAL  OF  BACTERIOLOGY 


Other  Diphtheria-like  Bacilli 

As  already  mentioned,  diphtheria-like  bacilli  are  not  infrequent 
in  wounds,  pathological  discharges  and  secretions.  Some  of  them 
may  be  positive  with  Neisser's  stain.  They  are  always  non-virulent. 
The  fermentation  reactions  of  some  of  these  organisms  will  be  found 
in  the  Table  on  p.  292. 


Bacillus  diphtherias  columbarum 

Pigeon  diphtheria  is  an  infectious  disease  of  pigeons,  charac- 
terised by  the  formation  of  diphtheritic -like  membranes  on  the 
tongue,  fauces,  and  corners  of  the  mouth  ;  occurs  in  extensive 
epizootics  from  time  to  time.  Loffler  isolated  a  bacillus  to  which 
he  gave  this  name.  It  is  short,  with  rounded  ends,  non-motile,  does 
not  form  spores,  and  does  not  stain  by  Gram's  method.  On  gelatin 
it  forms  a  whitish  growth  without  liquefaction,  on  agar  a  creamy 
growth,  and  on  potato  a  thin  grey  film.  Milk  is  not  curdled  and  is 
unchanged  in  reaction.  It  is  pathogenic  for  the  mouse  and  pigeon, 
but  only  slightly  so  for  the  fowl  and  guinea-pig.  It  is  possible  to 
prepare  a  vaccine,  and  an  anti-serum  for  the  disease.1  Recent 
research  has,  however,  suggested  that  the  disease  may  be  due  to  a 
filter-passer. 2 

Diphtheritic  roup  of  poultry  is  a  different  disease,  and  is  stated 
to  be  due  to  a  protozoan  parasite.3  Macfadyen  and  the  writer  4 
found  Klebs-Loffler-like  organisms  to  be  present  in  the  mouths  and 
throats  of  healthy  pigeons  and  fowls.  These  organisms  resembled 
the  true  Klebs -Loffler  bacillus  in  their  cultural  reactions,  but  were 
quite  non-virulent  to  guinea-pigs  (see  Table,  p.  292). 

The  so-called  diphtheria  of  calves  is  produced  by  an  anaerobic 
streptothrix. 

1  See  Ann.  de  rinst.  Pasteur,  xv,  1901,  p.  952. 

2  Dean  and  Marshall,  Journ.  of  Path,  and  Bact.,  vol.  xiii,  1908,  p.  29. 

3  See  also  Gordon  Sharp,  Lancet,  1900,  vol.  ii,  p.  18. 

4  Trans.  Path.  Soc.  Lond.,  vol.  li,  1900,  p.  13,  and  Brit.  Med.  Journ., 
1900,  vol.  i,  p.  994. 


CHAPTER  IX 

"  ACID-FAST  "  BACILLI 

TUBERCULOSIS— LEPROSY— THE  SMEGMA  BACILLUS- 
GLANDERS 

11  Acid-fast  "  Bacilli 

AN  important  characteristic  of  the  tubercle,  leprosy,  smegma,  and 
certain  other  bacilli  is  the  property  they  possess  when  stained 
with  fuchsin  of  retaining  the  red  colour  after  treatment  with  a 
strong  solution  of  a  mineral  acid  (25  per  cent,  sulphuric  or  30  per 
cent,  nitric).  They  are  therefore  termed  "  acid-fast."  Most  other 
organisms  are  rapidly  decolorised  even  by  1  or  2  per  cent,  sulphuric 
acid,  but  it  must  be  recognised  that  several  apparently  saprophytic 
bacilli  are  also  "  acid-fast."  The  retention  of  the  fuchsin  colour  in 
spite  of  treatment  with  the  acid  seems  to  be  due  to  the  presence  of 
substances  of  a  fatty  or  waxy  nature  within  the  organisms  with  which 
the  fuchsin  either  combines  or  is  protected  from  the  action  of  the  acid. 

Moreover,  by  cultivating  many  saprophytic  bacilli  in  media 
containing  butter,  Bienstock  and  Gottstein  converted  them  into 
"  acid -fast  "  forms. 

"  Acid-fast  "  bacilli  are  also  present  in  Johne's  disease,  occasion- 
ally in  rats,  in  butter  (Petri,  Rabinowitsch,  Rubner),  on  certain 
Graminaceae  (the  "  Timothy -grass  bacillus  "  of  Moeller),  and  in 
dung  (the  "  Mist  bacillus  ").  It  has  been  suggested  that  these 
saprophytic  acid-fast  bacilli  may  be  derived  from  the  tubercle 
bacillus,  but  Panisset's  work  gives  no  confirmation  of  this. 

The  StreptotricheaB  occasionally  exhibit  "  acid-fast  "  properties. 
All  the  acid-fast  bacilli  seem  to  be  Gram-positive. 

Tuberculosis 

Tuberculosis  is,  unfortunately,  only  too  common  in  the 
human  subject,  and  most  of  the  domestic  animals  and  wild 
animals  in  a  state  of  captivity  may  be  attacked  by  it. 

299 


300  A  MANUAL  OF  BACTERIOLOGY 

The  conception  of  tuberculosis  was  originally  a  purely 
anatomical  one,  the  name  being  given  to  a  condition  in 
which  the  organs  were  studded  with  little  yellowish  points 
or  nodules,  which  were  termed  tubercles.  Laennec  was 
the  first  to  indicate  the  characters  of  these  nodules  or 
tubercles,  and  traced  with  considerable  accuracy  their 
development  from  minute  lesions,  the  miliary  tubercles, 
up  to  the  large  cheesy  masses  which  may  be  met  with  in 
the  glands  and  lungs. 

Microscopically,  the  structure  of  a  young  and  typical 
tubercle  is  characteristic.  At  the  centre  one  or  more  giant- 
cells  are  found — large  protoplasmic  masses,  each  containing 
ten  to  twenty  nuclei  arranged  round  the  periphery  (Plate 
IX.  6).  They  are  of  the  nature  of  plasmodia,  similar  to 
the  masses  of  fused  cells  which  surround  a  foreign  body  in 
the  lower  animals  (Adami).  Around  the  giant- cells  are 
well-defined  epithelial- like  cells  with  large  and  distinct 
nuclei,  which  are  known  as  epithelioid,  or  more  properly 
endothelioid,  cells.  A  zone  of  smaller  cells  with  scanty 
protoplasm  and  small  nuclei  surrounds  the  endothelioid 
cells  ;  they  are  known  as  lymphoid  cells  from  their  likeness 
to  the  cells  of  lymphoid  tissue.  This  is  the  structure  of  a 
typical  tubercle,  but  one  or  other  of  the  components  may 
be  wanting,  and  none  can  be  said  to  be  absolutely  charac- 
teristic of  the  tubercle.  The  nodule  possesses  no  blood- 
vessels, and  as  its  size  increases  by  growth  at  the  periphery 
the  central  parts  undergo  degenerative  changes,  and  may 
become  either  structureless  or  hyaline,  or  be  converted 
into  a  soft  yellowish  material  somewhat  like  cheese  and 
termed  caseous.  More  or  less  extensive  inflammatory 
reaction  ensues  in  the  tissues  surrounding  the  tubercle, 
and  the  cellular  elements  so  produced  often  become  spindle- 
shaped  and  ultimately  fibrous,  so  that  the  tuberculous 
nodule  becomes  enclosed  by  a  capsule  of  fibrous  tissue 
which  may  contract  and  convert  it  into  a  fibrous  nodule. 


THE  TUBERCLE  BACILLUS  301 

After  caseation  has  occurred  calcification  may  ensue — 
that  is,  lime-salts  are  deposited  and  the  nodule  is  converted 
into  a  calcareous  mass. 

So  far  back  as  1865  Villemin  showed  that  inoculation 
of  rabbits  with  human  caseous  material  was  followed  by  a 
development  of  nodules  similar  in  all  respects  to  the  miliary 
tubercles  in  man.  Cohnheim,  Burdon  Sanderson,  and 
Wilson  Fox  confirmed  this  observation,  but  they  also 
showed  that  the  development  of  tubercles  apparently 
followed  the  introduction,  not  only  of  tuberculous  material, 
but  also  of  setons,  pieces  of  putrid  muscle,  and  gutta- 
percha.  It  was  pointed  out,  however,  that  in  all  proba- 
bility these  results  were  due  to  accidental  contamination 
or  inoculation  with  tuberculous  matter,  and,  by  adopting 
suitable  precautions  in  order  to  prevent  such  sources  of 
error,  it  was  conclusively  shown  that  non-tuberculous 
matter  is  unable  to  set  up  tuberculosis.  Tuberculosis  is 
therefore  inoculable,  and  is  an  infective  disease,  and  as 
such  must  be  due  to  a  specific  infective  agent,  to  the 
discovery  of  which  observers  then  directed  their  attention. 
In  1882  Koch  announced  that  he  had  discovered  a  special 
bacillus,  the  tubercle  bacillus,  in  tuberculous  tissues,  which 
could  be  isolated  and  cultivated,  and  which  reproduced  the 
disease  on  inoculation. 


The  Tubercle  Bacillus 

Morphology. — The  tubercle  bacillus  (B.  tuberculosis)  is 
a  slender  rod  with  rounded  ends,  often  slightly  curved,  and 
averaging  2-3  /m  in  length,  though  the  length  varies  in 
the  tissues  from  1*25  /JL  to  6'5  JUL  ;  in  cultures  it  tends  to  be 
short,  on  serum  being  about  1  yu.  In  stained  preparations 
one  or  more  unstained  intervals  are  often  seen  in  the  rods 
(Plate  VIII.  a) ;  these  have  been  considered  by  some 
observers  to  be  spores,  but  there  are  many  objections  to 


302  A  MANUAL  OF  BACTERIOLOGY 

this  view.  Spores  are  usually  single  and  not  multiple, 
and  are  regular  spherical  or  ovoid  bodies,  whereas  the 
unstained  spaces  in  the  tubercle  rods  are  irregular.  More- 
over, in  the  same  specimen  of  sputum  a  varying  amount 
of  "  beading,"  as  it  is  termed,  may  be  brought  out  by 
different  staining  methods  (Plate  VIII.  6)  ;  'in  a  prepara- 
tion stained  by  Gram's  method  it  is  usually  more  pro- 
nounced than  in  one  stained  with  carbol-fuchsin.  In  class 
work  also  it  will  be  found  that  one  student's  specimen  will 
show  beading  much  more  markedly  than  another's.  These 
considerations  render  it  probable  that  the  beading  is  partly 
due  to  segmentation  of  the  protoplasm,  and  partly,  perhaps, 
is  an  artifact  due  to  the  staining  process,  and  is  not  a 
spore  formation.  The  tubercle  bacillus,  however,  probably 
does  form  spores,  though  this  is  a  debated  point.  Some 
observers  have  described  clear,  regular,  unstained  spaces 
in  bacilli  from  old  cultivations,  and  consider  these  to  be 
true  spores. 

The  tubercle  bacillus  is  a  non-motile,  strictly  parasitic 
organism  (it  has  been  described  as  being  both  motile  and 
flagellated).  It  usually  occurs  singly,  occasionally  linked 
in  twos  or  threes  so  as  to  form  short  chains,  and  under 
certain  conditions,  especially  in  old  cultures,  filamentous 
forms  develop,  and  Foulerton1  and  others  include  it 
among  the  Streptotrichece.  The  bacillus  is  agglutinated 
by  the  blood-serum  of  a  tuberculous  animal  (see  p.  324). 
There  are  several  varieties  of  the  tubercle  bacillus  (see 
pp.  315  and  319). 

Staining  reactions. — The  tubercle  bacillus  stains  in- 
differently with  the  ordinary  watery  solutions  of  dyes, 
prolonged  treatment  with,  or  warming,  the  solution  being 
required.  It  stains  well  by  Gram's  method.  It  also 
stains  well  and  deeply  with  carbol-fuchsin,  particularly  on 
warming,  and  when  so  stained  is  markedly  resistant  to 
1  "  Milroy  Lectures,"  Lancet,  1910,  vol.  i,  p.  551,  et  seq. 


PLATE  VIII. 


<^  - 


v?mfi  "s*i 


The  tubercle  bacillus.     Film  preparation  of  a  pure 
culture,      x  1000. 


b.  Tubercle  bacilli  in  sputum,      x  1500. 


THE  TUBERCLE  BACILLUS  303 

the  decolorising  action  of  25-30  per  cent,  mineral  acid  ; 
that  is  to  say,  it  is  strongly  "  acid-fast,"  and  this  property 
is  made  use  of  for  demonstrating  its  presence  in  tissues, 
etc.,  and  for  diagnostic  purposes.  This  "  acid-fastness  " 
is  due  to  the  chemical  constitution  of  the  bacillus  (see 
p.  309).  In  old  and  particularly  healing  lesions  red- 
staining  granules  may  take  the  place  of  definite  bacilli : 
these  are  the  "  splitter  "  forms  of  Spengler. 

Cultural  characters. — The  tubercle  bacillus  is  aerobic 
and  facultatively  anaerobic,  and  thrives  best  at  a  tem- 
perature of  37°  C.  or  thereabouts,  but  development  even 
then  is  slow,  four  weeks  at  least  being  required  for  an 
appreciable  growth.  Primary  cultivations  from  the  lesions 
cannot  be  obtained  on  ordinary  culture  media  but  should 
be  made  on  (a)  Dorset's  egg  medium,  (6)  glycerinated 
potato  in  Roux's  tubes  (Fig.  9),  the  bulb  being  filled 
with  5  per  cent,  glycerin  in  physiological  salt  solution, 
(c)  glycerin  brain  agar,  or  (d)  glycerinated  serum  (preferably 
dogs').  Twort1  has  successfully  isolated  the  bacillus 
from  sputum  by  direct  cultures  in  an  ericolin  medium. 
Dorset's  egg  medium  is  prepared  thus  :  the  contents  of 
four  eggs  are  well  beaten  up,  25  c.c.  of  water  are  added, 
and  the  mixture  is  strained  through  muslin.  The  fluid  is 
then  tubed,  and  the  tubes  are  heated  in  the  sloping  position 
to  70°  C.  for  four  hours.  At  the  time  of  inoculation,  a 
drop  or  two  of  sterile  water  should  be  added.  Brain  agar 
is  prepared  by  making  a  3  per  cent,  nutrient  agar  of  -f  20 
reaction,  adding  an  equal  volume  of  pounded  ox-brain, 
and  sufficient  glycerin  to  make  5  per  cent,  in  the  mixture, 
and  sterilising.  Egg  broth  is  also  a  good  culture  medium. 

After  culture  on  these  media  for  some  generations,  the 
tubercle  bacillus  will  develop  on  5  per  cent,  glycerin  agar 
(reaction  +  15  or  20),  and  in  5  per  cent,  glycerin  broth 
(veal  is  best)  ;  it  will  also  grow,  though  very  slowly,  on 

1  Proc.  Roy.  Soc.  Lond.,  B   vol.  Ixxxi,  1909. 


304  A  MANUAL  OF  BACTERIOLOGY 

glycerin  gelatin  at  22°  C.  Gelatin  and  blood-serum  are  not 
liquefied.  On  glycerin  agar  the  growth  forms  a  dry,  crinkled 
and  wrinkled,  cream-coloured  or  brownish-yellow  film,  which 
has  been  well  described  as  resembling  the  patches  of  lichen 
met  with  on  trees  (Fig.  37).  The  growth, 
however,  varies  considerably,  both  in 
colour  and  in  the  amount  of  wrinkling, 
though  retaining  more  or  less  the  char- 
acteristics just  mentioned.  In  broth  it 
forms  soft,  cream-coloured,  flaky  masses, 
which  increase  slowly  both  in  size  and 
number,  the  broth  remaining  perfectly 
bright  and  clear.  Sometimes  a  dry  crink- 
led film  forms  on  the  surface  of  the  broth, 
and  may  spread  all  over  it,  and  tends  to 
creep  up  the  sides  of  the  vessel.  This  film 
formation  seems  to  be  essential  for  the 
preparation  of  a  satisfactory  old  tuber- 
culin, but  it  is  necessary  in  order  to  start 
it  that  some  of  the  inoculated  particles 
should  float  and  form  nuclei  from  which 
FIG.  37.  — Tubercle  the  film  spreads.  The  virulent  organism 
bacillus  Glycerin-  from  the  primary  cultivations  is  difficult 

agar  culture  three  ,  . 

months  old.  to  grow  on  anything  but  glycermated 

potato  or  serum,  or  brain  agar. 

TUBERCULINS. — Extracts  of,  and  suspensions  of  tritu- 
rated, tubercle  bacilli,  human  or  bovine,  are  employed 
in  treatment  and  for  the  diagnosis  of  tuberculosis.  The 
preparations  are  known  as  tuberculins. 

Old  tuberculin,  T.A. — This  is  prepared  by  growing  the 
tubercle  bacillus  in  glycerin  veal  broth  in  a  shallow  layer 
in  flat  flasks  (Fig.  38),  so  that  there  is  a  free  supply  of 
oxygen.  After  some  weeks  an  abundant  growth  with 
copious  film  formation  develops  ;  the  latter  seems  to  be 
essential,  but  it  does  not  appear  to  matter  whether  the 


OLD  TUBERCULIN  305 

bacilli  be  virulent  or  non-virulent,  or  whether  they  be 
of  human  or  of  mammalian  origin.  The  cultures,  bacilli 
and  all,  are  heated  at  115°  C.  in  the  autoclave  for  half  an 
hour,  then  concentrated  over  a  water-bath  to  about  one 
tenth  of  their  volume,  and  finally  are  filtered  through 
porous  porcelain  ;  the  resulting  fluid  is  thick,  owing  to 
the  concentration  of  the  glycerin  by  the  evaporation,  is 
of  a  dark  amber  colour,  and  possesses  a  curious  charac- 


FIG.  38. — Flask  for  growing  tuberculin. 

teristic  smell.     The  large  proportion  of  glycerin  preserves 
the  fluid,  which  keeps  indefinitely  in  a  cool  dark  place. 

This  old  tuberculin  possesses  remarkable  properties. 
Relatively  large  amounts  (O1-O5  c.c.)  may  be  injected 
into  a  healthy  animal  or  individual  without  effect,  but 
in  a  tuberculous  one  a  minute  dose,  O001  c.c.  or  less,  gives 
rise  to  a  marked  reaction — elevation  of  temperature  with 
constitutional  disturbance  more  or  less  severe,  and  swelling 
and  tumefaction  of  tuberculous  lesions  (glands,  ulcers, 
etc.),  and  this  reaction  is  made  use  of  for  diagnostic  pur- 
poses (see  p.  330).  By  cautiously  increasing  the  amount 
a  toleration  is  gradually  induced,  so  that  considerable 
doses  cause  little  or  no  disturbance.  Injections  of  tuber- 
culin tend  to  produce  marked  changes  in  the  tuberculous 
parts,  leading  to  necrosis  and  exfoliation,  with  subsequent 
healthy  reaction  and  repair.  This  is  especially  seen  in 
cases  of  lupus  ;  by  continued  injections  a  marvellous 

20 


306  A  MANUAL  OF  BACTERIOLOGY 

improvement  results,  so  much  so  that  a  cure  is  apparently 
effected ;  but,  unfortunately,  when  the  treatment  is 
discontinued  the  scar  usually  breaks  down  and  the  disease 
returns.  Nevertheless,  a  few  cases  have  remained  perma- 
nently healed. 

For  treatment,  the  dose  to  commence  with  should  not 
be  more  than  O0001  c.c.,  dilutions  being  made  with  O5 
per  cent,  carbolic  solution,  and  the  dose  is  repeated  when 
all  reaction  has  passed  away  and  is  gradually  increased. 
Tuberculin  R,  or  tuberculin  BE,  is  now  generally  employed 
(see  below). 

Healthy  guinea-pigs  bear  considerable  injections  of 
tuberculin  without  harm  ;  but  if  they  be  tuberculous,  if 
the  disease  is  advanced  (eight  to  ten  weeks  after  inocula- 
tion), doses  of  O01  c.c.  produce  death  ;  if  less  advanced 
(four  to  five  weeks  after  inoculation)  a  larger  dose,  O2 
to  O3  c.c.,  is  required  ;  but  O5  c.c.  always  proves  fatal. 
The  post-mortem  appearances  are  congestion  of  the 
lymphatics  and  viscera,  and  dark  red  spots,  from  mere 
points  to  the  size  of  a  hemp-seed,  on  the  liver  and  spleen. 
These  are  due  to  enormous  engorgement  of  the  capillaries 
in  the  immediate  neighbourhood  of  tuberculous  deposits, 
actual  extravasations  of  blood  being  rarely  found.  The 
hsemorrhagic-like  spots  on  the  liver  are  almost  pathogno- 
monic  of  death  from  tuberculin. 

Absolute  alcohol  precipitates  the  active  principle  of 
tuberculin  in  the  form  of  a  whitish  flocculent  precipitate 
which  chemically  consists  of  proteoses.  This  precipitate, 
re-dissolved,  is  made  use  of  in  the  ophthalmic  reaction 
(p.  330).  Tuberculin  applied  to  the  scarified  skin  also 
gives  a  cutaneous  reaction  in  tuberculosis  (p.  330). 

Tuberculin  R,  or  TR,  new  tuberculin,  is  prepared  from 
young  and  virulent  cultures  of  the  tubercle  bacillus.  The 
growth  is  collected,  dried  in  vacuo,  and  triturated  by 
machinery.  Of  the  triturated  material,  1  grm.  is  treated 


TUBERCULINS  307 

with  100  c.c.  of  distilled  water,  and  centrifuged.  The 
supernatant  liquid  is  rejected,  and  the  residue  is  collected, 
dried,  again  triturated  and  centrifuged.  The  supernatant 
liquid  is  carefully  pipetted  off  and  kept,  while  the  residue 
is  again  submitted  to  the  same  treatment,  and  the  process 
is  repeated  until  no  solid  residue  is  left.  The  fluids  are 
then  mixed,  the  solid  content  is  estimated  gravimetrically, 
some  glycerin  is  added,  and  the  liquid  is  diluted  to  the 
correct  volume,  so  as  to  contain  2  mgrm.  of  solid  matter 
per  cubic  centimetre  (not  10  mgrm.  as  formerly  stated), 
and  for  use  is  diluted  with  20  per  cent,  sterile  glycerin 
solution. 

Tuberculin  R,  according  to  Koch,  possesses  distinct 
immunising  properties,  and  causes  neither  reaction  nor 
suppuration. 

For  treatment  of  tuberculosis  in  man  the  initial  dose  is 
equivalent  to  not  more  than  TTT oV o o  ~  TTTO TOO  ~  5 uW 
mgrm.  of  solid  matter,  according  to  the  nature  of  the  case. 
The  doses  are  given  subcutaneously  at  intervals  of  ten 
to  fourteen  days,  and  the  treatment  may  be  controlled 
in  the  earlier  stages  by  opsonic  determinations.  According 
to  Latham,  tuberculin  may  also  be  given  by  the  mouth. 
Cases  of  cutaneous  or  localised  tuberculosis,  and  those  in 
which  the  opsonic  index  to  tubercle  is  moderately  reduced, 
react  best. 

Tuberculin,  bacillary  emulsion  (BE),  is  an  emulsion  of 
the  powdered  bodies  of  tubercle  bacilli  in  50  per  cent, 
aqueous  glycerin.  The  mixture  is  allowed  to  sediment 
until  all  heavy  particles  have  deposited,  the  milky  super- 
natant fluid  is  pipetted  off,  and  standardised  so  as  to 
contain  5  mgrm.  of  solid  matter  per  c.c.  The  dosage  is 
similar  to  that  of  tuberculin  R. 

Behring  has  prepared  another  tuberculin,  tulase  or  TC, 
by  treating  tubercle  bacilli  with  chloral,  which  he  states 
has  a  marked  curative  action,  and  is  better  administered 


308  A  MANUAL  OF  BACTERIOLOGY 

by  the  mouth  than  by  subcutaneous  inoculation.  By 
giving  tulase  to  cows,  the  milk  is  said  to  acquire  immu- 
nising and  curative  properties  which  are  transmitted  to 
those  consuming  it.  Rosenbach's  tuberculin  is  prepared 
by  growing  the  tubercle  bacillus  with  the  ringworm  or- 
ganism, Friedmann's  is  derived  from  a  turtle  tubercle 
bacillus.  Other  tuberculins  are  also  on  the  market,  and 
any  tuberculin  may  be  prepared  with  a  human  or  with  a 
bovine  strain  of  bacillus. 

Chemical  products. — The  tubercle  bacillus  produces  no 
extra-cellular  toxin.  Crookshank  and  Herroun  obtained 
from  glycerin  broth  cultures  of  the  tubercle  bacillus  a 
proteose  and  an  alkaloidal  body.  The  proteose  was  also 
obtained  from  "  perlsucht."  Both  the  alkaloid  and  the  pro- 
teose (from  both  sources)  produced  a  rise  of  temperature  in 
tuberculous  guinea-pigs,  while  in  healthy  animals  the  former 
caused  a  slight,  and  the  latter  a  marked,  fall  in  temperature. 

De  Schweinitz  and  Dorset x  described  chemical  products 
isolated  from  the  tubercle  bacillus  grown  in  a  special 
glycerin-asparagin  mixture.  From  the  bacilli  themselves 
an  acid  body  was  isolated,  probably  teraconic  acid,  an 
unsaturated  acid  of  the  fatty  series.  A  certain  amount 
of  the  same  body  was  also  obtained  from  the  special  culture 
medium,  but  only  a  trace  from  glycerin  broth,  in  which 
the  bacilli  had  been  cultivated,  in  the  latter  case  not 
because  it  was  not  formed,  but  because  of  the  difficulty 
of  isolation.  This  acid  seemed  to  produce  on  injection 
depression  of  temperature  and  necrosis  of  the  tissues 
locally,  possessed  some  immunising  power,  and  may  be 
the  substance  producing  caseation  in  the  tuberculous 
nodules.  The  bacilli  extracted  with  hot  water  yielded  an 
albuminoid,  which  gave  the  tuberculin  reaction.  This 
they  regard  as  the  fever-producing  substance. 

1  Med.  Journ.  N.  Y.,  1897,  July  24,  p.  195.  Also  Fifteenth  Annual 
Rep.  Bureau  of  Animal  Industry,  U.S. A,,  189C 


ACTION  OF  HEAT  309 

Bulloch  and  Macleod  l  state  that  the  acid-fast  substance 
of  the  tubercle  bacillus  is  an  alcohol.  Hot  xylol  will 
remove  this  substance  from  the  tubercle  bacillus,  and 
ether  or  5  per  cent,  caustic  soda  that  from  the  smegma 
bacillus  ;  the  organisms  after  this  treatment  are  no  longer 
"  acid-fast." 

Maragliano  states  that  toxic  bodies  are  present  in  the 
blood  and  urine  of  tuberculous  individuals.  Cellulose  also 
seems  to  be  present  in  small  amount  in  the  bacilli  (it  has 
also  been  found  in  tuberculous  nodules). 

Tubercle  bacilli,  living  or  dead,  are  with  great  difficulty 
absorbed  when  in  any  quantity.  The  dead  bacilli  when 
injected  under  the  skin  invariably  cause  suppuration,  and 
several  months  later  it  is  still  possible  to  detect  in  the  pus 
numerous  bacilli  which  stain  well ;  introduced  into  the 
circulation  of  rabbits  they  give  rise  to  nodules  in  the  lungs 
similar  to  the  tuberculous  nodules  produced  by  living 
bacilli  (Koch). 

Action  of  heat  and  antiseptics  on  the  tubercle  bacillus.— 
The  thermal  death-point  of  the  bacillus  has  been  the 
subject  of  some  controversy.  Sternberg  found  that  tuber- 
culous sputum  exposed  for  ten  minutes  to  a  temperature 
of  90°,  80°,  and  66°  C.  failed  to  infect  guinea-pigs  in  inocu- 
lation, while  another  specimen  of  the  same  sputum  heated 
for  ten  minutes  to  a  temperature  of  50°  C.  produced  tuber- 
culosis in  a  guinea-pig,  so  that  from  these  experiments  the 
thermal  death-point  lies  between  50°  and  66°  C. 

Yersin  in  1888,  by  culture  methods,  failed  to  obtain 
any  growth  from  bacilli  which  had  been  heated  to  70°  C. 
for  ten  minutes,  while  those  heated  to  55°  C.  and  60°  C. 
gave  growths  in  glycerin  broth  in  ten  days  and  twenty-two 
days  respectively.  Macfadyen  and  the  writer,  in  the 
course  of  some  experiments  on  the  sterilisation  of  milk 
found  that  milk  to  which  powdered  dried  sputum  had  been 

1  Journ.  of  Hygiene,  vol.  iv,  1904,  p.  1. 


310  A  MANUAL  OF  BACTERIOLOGY 

added  was  rendered  innocuous  by  a  momentary  heating 
to  67°-68°  C.     These  experiments  indicate  that  a  tem- 
perature of  65°  C.  and  over  is  probably  rapidly  fatal  to 
the  tubercle  bacillus,  so  that  milk  which  has  been  pas- 
teurised (i.e.  heated  to  68°-70°  C.  for  twenty  to  thirty 
minutes)   may  be  regarded  as  quite  safe.     Experiments 
by  the  Royal  Commission  on  Tuberculosis  with  virulent 
tuberculous   milk   gave   somewhat   irregular   results ;     in 
one  instance  heating  to  65°  C.  for  two  and  a  half  minutes 
rendered  the  milk  innocuous,  in  another  instance  after  five 
minutes  at  70°  C.  it  was  slightly  virulent,  but  twelve  minutes 
at  the  same  temperature  rendered  it  inert  (see  also  section 
on  "  Milk  ").     Foulerton  found  that  emulsified  tuberculous 
material  from  tuberculous  guinea-pigs  did  not  lose  its  power 
of  infecting  unless  heated  to  70°  C.  or  over  for  ten  minutes. 
The  tubercle  bacillus  offers  considerable  resistance  to 
the  action  of  antiseptics  and  germicides.     Yersin  found 
that  it  was  killed  by  5  per  cent,  carbolic  acid  in  thirty 
seconds,  by  1  per  cent,  in  one  minute,  by  absolute  alcohol 
in  five  minutes,  and  by  mercuric  chloride,  1-1000,  in  ten 
minutes.     Crookshank    found    that    tuberculous    sputum 
mixed  with  an  equal  volume  of  5  per  cent,  carbolic  was 
rendered  innocuous  in  a  few  minutes,  and  this  without 
any  special  precautions  as  to  breaking  up  the   masses. 
For  disinfecting  sputum  mercuric  chloride  is  unsuitable. 
(See  also  Chap.  XXI.) 

Pathogenesis,  etc. — Man  is,  unfortunately,  only  too  fre- 
quently attacked  with  tuberculosis,  the  manifestations 
of  which  tend  to  differ  somewhat  at  different  age  periods. 
Thus,  in  the  very  young,  general  miliary  tuberculosis, 
tuberculous  meningitis,  and  tuberculous  disease  of  the 
peritoneum,  intestine,  and  mesenteric  glands  (tabes  mesen- 
terica)  are  the  commonest ;  in  older  children,  up  to  the 
age  of  puberty,  the  lymphatic  glands,  especially  in  the 
neck,  joints  and  bones,  and  the  skin  (lupus)  are  mostly 


PLATE  IX. 


a.  Tubercle  bacilli  in  sputum,      x  1000. 


i 


*v 

•  <^k    •  ..r 


*•%     ^r 


il+  t 


«    •  ^  & 


&.  Giant-cell  in  a  tubercle  containing  tubercle  bacilli,      x  1000. 


DISTRIBUTION  OF  BACILLI  311 

attacked  ;  young  adults  suffer  from  disease  of  the  lung 
(consumption,  phthisis),  and  older  people  from  chronic 
disease  of  the  lung  and  tuberculous  disease  of  the  urinary 
organs  and  testes,  and  of  the  suprarenal  capsules  (Addi- 
son's  disease).  Scrofula  and  struma  were  terms  formerly 
much  employed  ;  both  denote  a  swollen  neck,  and  were 
applied  to  cases  suffering  from  chronic  tuberculous  inflam- 
mation with  enlargement  of  lymphatic,  especially  of  the 
cervical,  glands,  with  which  other  conditions,  such  as 
inflammations  of  the  ear,  throat  and  eye,  and  implication 
of  bones  and  joints,  are  frequently  associated. 

The  distribution  of  the  bacillus  in  the  tissues  varies 
considerably.  In  young  and  active  tubercles  the  bacilli 
are  more  plentiful  and  more  easily  demonstrated  than  in 
older  and  more  chronic  ones.  They  tend  to  be  more 
numerous  in  some  animals  than  in  others — in  the  ox  and 
horse  than  in  man,  for  example.  In  man  the  bacillus  is 
difficult  to  demonstrate  (by  staining)  in  enlarged  and 
caseating  glands,  in  pus,  in  synovial  membranes,  and  in 
lupus.  In  some  animals,  especially  the  ox  and  horse, 
bacilli  can  usually  be  readily  demonstrated,  and  may  be 
present  in  large  numbers,  and  frequently  have  the  typical 
distribution,  viz.  within  and  at  the  periphery  of  the  giant- 
cells,  though  they  are  by  no  means  confined  to  this  locality 
(Plate  IX.  b). 

It  was  asserted,  particularly  by  Rosenberger  and  For- 
syth,  that  tubercle  bacilli  can  be  detected  in  the  blood  in 
the  majority  of  cases  of  pulmonary  tuberculosis.  Hewat 
and  Sutherland,1  however,  made  twenty-two  blood  exami- 
nations on  twenty  patients  in  all  stages  of  the  disease  and 
in  only  one  detected  two  acid-fast  bacilli.  Schroeder  and 
Cotton  tested  the  blood  of  forty-two  cattle  in  all  stages  of 
tuberculosis  by  inoculation  into  guinea-pigs  with  negative 
results. 

1  Brit.  Med.  Journ.,  1909,  vol.  ii,  p.  1119  (References). 


312  A  MANUAL  OF  BACTERIOLOGY 

Tuberculosis  in  animals. — The  majority  of  the  domestic 
animals  are  subject  to  tuberculosis.  It  is  most  common 
in  the  ox,  pig,  and  horse,  much  less  so  in  the  sheep  and 
goat,  cat  and  dog.  Wild  animals,  both  mammals  and 
birds,  in  a  state  of  captivity  are  also  specially  prone  to  be 
attacked,  and  a  large  number  of  the  deaths  in  Zoological 
Gardens,  particularly  among  the  apes,  are  due  to  this 
disease. 

In  the  ox  the  tuberculous  lesions  are  most  frequently 
met  with  in  the  lymphatic  glands  and  serous  membranes, 
particularly  the  pleura,  and  in  the  lungs  and  liver,  while 
the  fat  and  muscular  tissues,  which  constitute  the  major 
part  of  "  meat,"  are  very  rarely  affected.  On  the  pleura 
the  growths  take  the  form  of  nodular  masses,  which  from 
their  arrangement  are  popularly  termed  "  grapes "  or 
"  angle  berries." 

In  carp,  tubercle-like  nodules  are  occasionally  met  with 
in  which  a  bacillus  resembling  the  tubercle  bacillus  in 
morphology  and  staining  reactions  is  present.  It  grows, 
however,  much  more  freely  than  the  true  tubercle  bacillus, 
and  though  inoculable  into  fish  and  frogs,  is  non-inoculable 
into  warm-blooded  animals.  But  it  yields  a  tuberculin 
which  reacts  with  mammalian  tuberculosis,  and  by  feeding 
carp  on  the  mammalian  tubercle  bacillus  this  can  apparently 
be  transformed  into  the  piscian  variety.1 

Bird  or  avian  tuberculosis  undoubtedly  differs  in  many 
respects  from  mammalian  tuberculosis.  The  tuberculous 
new  formations  may  be  very  large,  but  do  not  show  nearly 
such  a  disposition  to  caseation  or  suppuration  as  the 
human  lesions.  Epithelioid  cells  form  the  major  part  of 
the  growth,  and  giant-cells  are  very  infrequent.  One 
remarkable  feature  is  the  enormous  numbers  of  bacilli 
which  may  be  present  in  the  tissues  ;  in  places  they  may 
be  so  numerous  and  closely  packed  as  to  form  distinct 

1  See  Himmelberger,  Centr.  f.  Bakt.,  Abt.  I  (Orig.),  vol.  73,  p.  1. 


AVIAN  TUBERCULOSIS  313 

masses  or  nodules.  The  bacilli  of  avian  have  the  same 
staining  reaction  as  those  of  mammalian  tuberculosis,  but 
on  cultivation  and  inoculation  various  differences  between 
the  two  races  become  evident.  Rats,  guinea-pigs,  and 
rabbits  are  practically  insusceptible  to  inoculation  with 
the  avian  bacillus. 

The  mammalian  bacilli  flourish  best  at  about  37°  C., 
and  growth  ceases  at  41°  C.,  whereas  the  avian  bacilli 
thrive  luxuriantly  at  43°  C.,  and  the  growth  of  the  latter 
on  glycerin  agar  is  much  moister  and  more  wrinkled,  and 
often  more  pigmented,  than  that  of  the  former.  Fowls 
and  dogs  are  with  difficulty  infected  with  human  bacilli, 
but  dogs  are  susceptible  to  infection  with  avian  bacilli. 
By  cultivation  on  boric-acid  agar  and  on  eggs,  etc.,  the 
mammalian  bacilli  are  stated  to  assume  the  characters  of 
the  avian. 

Avian  tuberculosis  is  of  practical  importance  not  only 
as  attacking  poultry,  but  also  in  human  pathology,  as 
several  cases  have  been  recorded  in  which  the  bacilli 
cultivated  from  human  cases  seemed  to  be  of  the  avian 
type,  and  were  therefore  probably  derived  from  an  avian 
source  of  infection.  Two  types  of  tuberculosis  also  occur 
in  the  horse — one  in  which  the  lesions  are  chiefly  abdominal, 
in  the  other  the  lungs  and  bronchial  glands  are  most 
affected.  Nocard  states  that  the  bacillus  obtained  from 
the  pulmonary  variety  is  generally  of  the  ordinary  mam- 
malian type,  while  that  of  the  abdominal  one  belongs  to 
the  avian. 

Relation  of  human  and  bovine  tuberculosis. — It  was 
noticed  long  ago  that  there  are  certain  differences  between 
the  bacilli  of  human  and  of  bovine  tuberculosis,  the  latter 
tending  to  be  shorter  and  thicker  and  less  readily  culti- 
vated than  the  former  ;  also,  whereas  human  tuberculous 
material  injected  into  a  rabbit  generally  produces  small 
discrete  lesions  which  tend  to  retrogress,  bovine  material 


314  A  MANUAL  OF  BACTERIOLOGY 

induces  a  progressive  disease  with  large  caseating  masses.1 
These  distinctions  were  regarded  as  being  due  to  variations 
in  the  bacilli  as  a  result  of  growing  upon  a  different  soil 
and  not  to  any  fundamental  difference  between  the  two 
strains  of  bacilli.  In  1901,  however,  Koch  stated  2  that 
young  cattle  and  swine  cannot  be  infected  with  human 
tuberculous  material,  and  he  therefore  concluded  that 
human  and  mammalian  tubercle  bacilli  are  essentially 
different.  As  a  result  of  his  experiments  he  made  the 
statement  that  "  though  the  important  question  whether 
man  is  susceptible  to  bovine  tuberculosis  at  all  is  not  yet 
absolutely  decided,  if  such  a  susceptibility  really  exists, 
the  infection  of  human  beings  is  but  a  very  rare  occurrence." 
This  view  met  with  considerable  opposition,  and  a 
second  Royal  Commission  was  appointed  to  investigate 
the  question,  and  the  following  summarises  the  results 
obtained  up  to  the  present,  from  which  it  will  be  gathered 
that  while  there  is  no  justification  for  assuming  that  man 
is  infected  from  human  sources  alone,  infection  from 
human  sources  is  probably  vastly  more  frequent  than 
from  any  other.  Thirty  different  viruses  isolated  from 
cases  of  tuberculosis  occurring  spontaneously  in  bovines 
have  been  studied,  and  the  results  of  introducing  them 
into  a  number  of  different  animals  by  feeding  and  inocula- 
tion are  recorded.  In  calves,  inoculation  usually  results 
in  generalised  progressive  tuberculosis,  but  the  effect  is 
somewhat  dependent  on  the  dose,  i.e.  the  number  of  bacilli, 
administered.  Thus  whereas  50  mgrm.  of  culture  always 
induced  a  fatal  generalised  progressive  tuberculosis,  in 
two  instances  much  smaller  doses — 0'01-0'02  mgrm. — 
produced  only  limited  retrogressive  tuberculosis.  Feeding, 
on  the  other  hand,  usually  produced  lesions  limited  to 

1  The  bacill    derived  from  tuberculosis  of  the  sheep,  pig,  and  horse 
(pulmonary  lesions)  are  also  of  the  bovine  type. 

2  See  Brit.  Med.  Journ.,  1901,  vol.  ii,  p.  189. 


BOVINE  TUBERCULOSIS  315 

the  neighbourhood  of  the  digestive  tract,  which  generally 
retrogress  and  become  calcareous.     The  bovine  bacillus, 
when  introduced   into  rhesus  monkeys   or  chimpanzees, 
either  by  inoculation  (even  in  so  small  a  dose  as  0-001 
mgrm.)  or  by  feeding,  induces  rapid  generalised  tuberculosis, 
and,  considering  the  close  relation  that  exists  between  the 
anthropoid  apes  and  man,  these  results  are  of  the  highest 
importance.     In  pigs,  generalised  progressive  tuberculosis 
is  readily  set  up  both  by  feeding  with,  and  by  the  inocula- 
tion of,  bovine  bacilli.     Goats,  dogs,  and  cats  are  relatively 
less  susceptible,  but  more  or  less  tuberculous  infection  can 
similarly  be  produced  in  them.     On  this  part  of  the  inves- 
tigation the  Commissioners  remark  that  the  bacillus  of 
bovine  tuberculosis  is  not  so  constituted  as  to  act  on 
bovine  tissues  only,  and  the  fact  that  it  can  readily  infect 
the  anthropoid  apes,  and,  indeed,  seems  to  produce  this 
result  more  readily  than  in  the  bovine  body  itself,  has  an 
importance  so  obvious  that  it  need  not  be  dwelt  on.     The 
viruses  isolated  from  sixty  cases  of  the  disease  in  man 
were  also  studied,  and  the  results  obtained  show  that  they 
may  be  divided  into  two  groups,  subsequently  referred  to 
as  Group  I  and  Group  II.     The  bacilli  of  Group  I  com- 
prised fourteen  viruses,  one  obtained  from  sputum,  three 
from  tuberculous  cervical  glands,  and  ten  from  mesenteric 
glands    of   primary   abdominal   tuberculosis   in   children. 
The  results  produced  by  introducing  these  viruses  into 
animals  are  identical  with  those  produced  by  the  bovine 
bacillus.      The  bacilli  of  Group  II  comprised  forty  viruses 
obtained  from  various  forms  of  human  tuberculosis — cer- 
vical glands,  mesenteric  glands  (8),  lungs  and  bronchial 
glands  (10),  joint  and  bone  disease  (9),  testis,  kidney,  etc. — • 
grow  more  luxuriantly  in  culture  than  those  of   Group  I, 
and  inoculated  into  calves  and  rabbits  do  not  produce 
the  generalised  and  fatal  disease  caused  by  the  bovine 
bacillus,  but  in  rhesus  monkeys  and  in  the  chimpanzee 


316  A  MANUAL  OF  BACTERIOLOGY 

set  up  a  general  tuberculosis.  Certain  human  viruses, 
differing  in  certain  respects  from  those  of  Groups  I  and 
II,  were  also  met  with  and  are  classed  as  Group  III,  but 
an  opinion  on  their  significance  is  reserved  for  a  future 
report. 

The  Commissioners  conclude  that  the  tubercle  bacillus 
in  its  nutritive  and  reproductive  powers  resembles  other 
simple  organisms,  and  that  the  essential  difference  between 
one  strain  and  another  depends  on  variations  in  these 
factors,  and  they  classify  those  bacilli  that  grow  with 
difficulty  on  artificial  media  as  dysgonic,  and  those  that 
grow  readily  on  media  as  eugonic. 

As  regards  the  histological  appearances  of  the  tuber- 
culous process  in  different  animals,  Eastwood  states  that 
there  is  an  underlying  unity  of  the  morbid  processes 
produced  experimentally  by  infection  with  every  variety 
of  bovine  and  human  tubercle  bacillus. 

In  their  final  Report,  the  Commissioners  conclude  that 
an  appreciable  amount  of  human  tuberculosis  is  caused 
by  bacilli  of  the  bovine  type,  and  that  tuberculosis  may  be 
communicated  to  man  from  infected  cow's  milk,  and  from 
tuberculous  meat,  either  beef  or  pork. 

So  far,  therefore,  from  any  relaxation  of  the  existing 
supervision  of  milk-production  and  meat-preparation  being 
possible,  the  Commissioners  press  upon  the  Government 
the  enforcement  of  food  regulations,  "  planned  to  afford 
better  security  against  the  infection  of  human  beings 
through  the  medium  of  articles  of  diet  derived  from  tuber- 
culous animals."  More  particularly  they  urge  such  action 
"  in  order  to  avert  or  minimise  the  present  danger  arising 
from  the  consumption  of  infected  milk." 

Of  young  children  who  died  of  wasting  disease  of  the 
intestine,  the  bovine  bacillus  was  present  in  nearly  half 
the  cases.  Further,  a  large  proportion  of  cases  of  tuber- 
culous cervical  glands  in  both  children  and  adults  was 


CHANNELS  OF  INFECTION  317 

due  to  the  same  bacillus.  The  wording  of  the  report  is  : 
"  Whatever,  therefore,  may  be  the  animal  source  of  tuber- 
culosis in  adolescents  and  in  adult  man,  there  can  be  no 
doubt  that  a  considerable  proportion  of  the  tuberculosis 
affecting  children  is  of  bovine  origin,  more  particularly 
that  which  affects  primarily  the  abdominal  organs  and 
the  cervical  glands.  And  further,  there  can  be  no  doubt 
that  primary  abdominal  tuberculosis,  as  well  as  tubercu- 
losis of  the  cervical  glands,  is  commonly  due  to  ingestion 
of  tuberculous  infective  material.  The  evidence  which 
we  have  accumulated  goes  to  demonstrate  that  a  con- 
siderable amount  of  the  tuberculosis  of  childhood  is  to 
be  ascribed  to  infection  with  bacilli  of  the  bovine  type 
transmitted  to  children  in  meals  consisting  largely  of  the 
milk  of  the  cow. 

"  We  are  convinced  that  measures  for  securing  the 
prevention  of  ingestion  of  living  bovine  tubercle  bacilli 
with  milk  would  greatly  reduce  the  number  of  cases  of 
abdominal  and  cervical  gland  tuberculosis  in  children, 
and  that  such  measures  should  include  the  exclusion  from 
the  food  supply  of  the  milk  of  the  recognisably  tuberculous 
cow,  irrespective  of  the  site  of  the  disease,  whether  in  the 
udder  or  in  the  internal  organs." 

Eber,1  in  an  extended  investigation,  succeeded  in  infect- 
ing calves  from  three  cases  of  human  pulmonary  tuber- 
culosis. The  bacilli  isolated  from  the  human  material  were 
of  the  human  type,  but  after  passage  through  the  calf 
became  transformed  into  the  bovine  type.  He  affirms, 
therefore,  the  essential  identity  of  the  human  and  bovine 
types  of  tubercle  bacilli. 

With  regard  to  the  channel  of  infection  in  human  tuber- 
culosis opinions  differ.  Koch  insisted  that  inhalation  of 
air- borne  bacilli  derived  from  dried  human  sputum  is  the 
principal  source  of  infection ;  Von  Behring,  on  the  other 

1  Centr.f.  BakL,  Abt.  I  (Orig.),  lix,  1911,  p.  193. 


318  A  MANUAL  OF  BACTERIOLOGY 

hand,  expressed  the  opinion  that  tuberculous  milk  fed  to 
children  is  the  main  source  of  infection  both  of  children 
and  of  adults ;  in  the  latter  case  he  suggested  that  bacilli 
are  ingested  in  childhood  and  lie  dormant  for  years  before 
becoming  active. 

Calmette  similarly  believes  that  in  the  young  infection 
by  the  digestive  tract,  especially  by  tuberculous  milk,  is 
the  more  frequent,  and  attaches  little  or  no  importance  to 
dry  dust  containing  tubercle  bacilli  as  a  source  of  infection. 
Ravenel  considers  that  the  alimentary  tract,  particularly 
in  children,  is  a  frequent  portal  of  entry  for  the  tubercle 
bacillus,  which  he  believes  is  able  to  pass  through  an  intact 
mucous  membrane.  Of  sixty  cases  of  human  tuberculosis 
investigated  by  the  Royal  Commission  on  Tuberculosis, 
twenty- eight  possessed  clinical  histories  indicating  that 
in  them  the  bacillus  might  have  been  introduced  by  the 
alimentary  canal.  Eraser  has  also  directed  attention  to 
the  frequency  of  the  bovine  type  of  bacillus  in  the  tuber- 
culous lesions  of  bone  and  joints  in  children. 

Fliigge,  on  the  other  hand,  states  that  his  experiments 
show  that  tuberculosis  can  be  communicated  to  animals 
by  inhalation,  and  that  the  dose  of  bacilli  required  to 
infect  by  the  respiratory  tract  is  far  less  than  that  required 
to  infect  by  the  alimentary  canal.  The  mode  of  infection 
in  man  doubtless  varies,  and  he  believes  that  children 
may  be  infected  by  the  digestive  tract,  by  tuberculous  food, 
particularly  milk,  but  the  most  extensive  source  of  infection 
is  the  number  of  droplets  of  tuberculous  expectoration 
coughed  up  by  consumptives  ;  these  float  in  the  air  and 
serve  as  sources  of  infection  to  others.  Ribbert  and 
Schrotter,  also,  from  the  evidence  of  autopsies,  considered 
inhalation  as  the  chief  mode  of  infection  in  man. 

Bulloch,1  from  a  careful  survey  of  the  literature,  con- 
cludes that  pulmonary  tuberculosis  is  invariably  caused 
1  "  Horace  Dobell  Lecture,"  1910. 


COMMISSION  ON  TUBERCULOSIS  319 

by  bacilli  of  the  human  type,  and,  therefore,  is  presumably 
due  to  inhalation  of  human  bacilli. 

McFadyean,1  also,  from  a  critical  survey  of  the  experi- 
mental evidence,  concludes  that  (1)  inhalation  of  tubercle 
bacilli  suspended  in  the  air  is  a  very  certain  method  of 
infecting  susceptible  animals  ;  (2)  experimental  infection 
by  the  digestive  tract  is  comparatively  difficult  to  realise  ; 
(3)  inhalation  is  probably  the  commonest  natural  method 
of  infection,  both  in  man  and  in  animals ;  (4)  infection 
by  the  digestive  tract  can  be  inferred  only  when  the 
lesions  are  confined  to  the  abdomen.  He  finally  states 
that  "  the  whole  of  the  experimental  evidence  on  which 
the  theory  of  the  intestinal  origin  of  pulmonary  tuber- 
culosis in  man  was  built  up  has  been  swept  away." 

While  the  death-rate  per  1000  living  from  all  forms  of 
tuberculosis  is  about  1-64,  that  from  phthisis  is  1-14,  so 
that  the  greater  part  of  the  mortality  from  tuberculosis 
must  be  ascribed  to  infection  from  human  sources.  There 
still  remains  the  residuum  of  glandular,  abdominal,  bone 
and  joint  tuberculosis  which  is  ascribed  to  infection  with 
the  bovine  bacillus.  The  experiments  of  the  Royal 
Commission  on  Tuberculosis  favour  this  view,  but  an 
alternative  explanation  is  possible.  Thus  Spengler,  Klem- 
perer,  and  Baumgarten  from  direct  experiments  on  man 
assert  that  the  bovine  bacillus  is  not  pathogenic  to  man, 
and  Spengler  distinguishes  two  types  of  human  tubercle 
bacilli,  (a)  the  "  humanus  brevis,"  the  ordinary  human 
type,  and  (b)  the  "  humanus  longus  "  type.  The  latter 
is  very  like  the  bovine  bacillus  in  pathogenic  action,  but 
after  carefully  weighing  all  the  facts,  Spengler  considers 
the  true  "  bovinus  "  and  the  "  humanus  longus  "  types  are 
not  identical.  It  may  be  then  that  the  bacillus  found  in 
certain  human  lesions  and  considered  to  be  the  bovine 
variety  is  really  this  humanus  longus  variety.  Even 

1  Jaurn.  Roy.  Inst.  Public  Health,  vol.  xviii,  1910,  p.  705. 


320  A  MANUAL  OF  BACTERIOLOGY 

admitting  that  the  bovine  bacillus  does  infect  man,  it  by 
no  means  follows  that  all  such  cases  of  infection  are  derived 
from  a  bovine  source,  for  humans  might  infect  one  another 
with  the  bovine  bacillus  ;  this  possibility  never  seems  to 
be  considered. 

The  occurrence  of  tuberculosis  in  the  domestic  animals  raises 
points  of  practical  importance,  especially  the  occurrence  of  infection 
from  the  consumption  of  meat  and  milk  from  diseased  animals. 
There  can  be  no  doubt  that  the  carcase  of  an  animal  extensively 
affected  with  tuberculosis,  especially  if  wasting  has  occurred,  should 
be  condemned  as  unfit  for  food,  and  likewise  all  parts  in  which 
there  are  tuberculous  deposits.  But  it  becomes  an  important 
question  for  the  community,  financially  as  well  as  from  a  hygienic 
point  of  view,  as  to  the  method  of  procedure  with  the  meat  from  a 
beast  comparatively  slightly  affected  with  tuberculosis — an  enlarged 
gland  or  two,  and  a  few  nodules  on  the  pleura.  No  doubt  the  ideal 
method  in  such  a  case  is  the  condemnation  and  destruction  of 
the  whole  carcase,  be  the  amount  of  tubercle  ever  so  little  ;  but 
from  financial  considerations  this  procedure  is  hardly  practicable 
on  account  of  the  large  amount  that  would  have  to  be  paid  in 
compensation.  Experiment  has  demonstrated  that  the  tubercle 
bacilli  are  practically  confined  to  the  tuberculous  areas  and  are 
extremely  rarely  met  with  in  the  muscular  tissue,  and  these  portions, 
therefore,  it  might  seem,  could  be  eaten  with  impunity,  especially 
as  they  would  be  cooked  before  consumption.  As  regards  swine, 
however,  it  is  generally  held  that  tuberculosis  anywhere  condemns 
the  whole  carcase. 

The  report  of  the  first  Royal  Commission  on  Tuberculosis,  how- 
ever, indicated  two  dangers.  Firstly,  in  cutting  up  a  carcase  the 
butcher  will  most  likely  use  the  same  knife  throughout,  and  in 
this  way  may  infect  the  meat  with  tuberculous  matter  by  smearing 
with  the  knife.  Secondly,  cooking  cannot  be  depended  upon  to 
destroy  the  bacilli  unless  the  joints  are  under  6  Ib.  in  weight ;  when 
the  weight  is  above  this  the  temperature  in  the  interior  may  not 
rise  sufficiently  high.  Evidently  one  of  the  first  measures  to  be 
taken  is  the  abolition  of  private  slaughter-houses  and  the  establish- 
ment of  municipal  abattoirs  where  the  meat  would  have  to  be 
passed  by  competent  inspectors.  In  this  way  all  badly  affected 
carcases  would  be  condemned,  and  those  only  slightly  affected  could 
be  separately  dealt  with  and  special  precautions  taken  to  eliminate 
tuberculous  pieces,  etc. 


TUBERCULOUS  MILK  321 

Tuberculous  milk  also  raises  many  important  points.  Probably 
some  10-15  per  cent,  of  all  samples  are  infective  to  guinea-pigs,  but 
this  does  not  necessarily  indicate  that  this  proportion  would  be 
dangerous  to  man,  for  the  material  is  introduced  into  the  guinea- 
pigs  by  inoculation  after  concentration  by  centrifuging  (see  also 
section  on  "  Milk  ").  Tubercle  bacilli  are  present  in  milk  not  only 
when  the  udder  is  tuberculous,  but  also  when  the  cows  are  suffering 
from  tuberculosis  elsewhere  which  is  clinically  recognisable.  Thus, 
when  the  lungs  are  affected,  bacilli  are  disseminated  from  the  air- 
passages  and  also  by  the  faeces.  It  is  noteworthy  that  the  incidence 
of  abdominal  tuberculosis  in  young  children  occurs  just  when  cow's 
milk  is  the  staple  article  of  their  diet.  At  the  same  time  this 
incidence  does  not  seem  to  fall  on  those  who  consume  most  milk. 

Much  might  be  done  by  the  registration  of  all  dairy  premises, 
the  use  of  selected  cows,  the  elimination  of  all  tuberculous  animals, 
and  by  enforcing  the  inspection  of  dairy  cattle  by  competent 
inspectors  at  suitable  intervals.  The  notification  of  all  forms  of 
udder  disease  is  now  compulsory.  In  the  absence  of  inspection 
and  the  use  of  selected  cows,  treatment  of  milk  intended  for  the 
food  of  infants  and  young  children  by  pasteurisation  or  sterilisation 
has  been  recommended,  but  has  disadvantages  (see  section  on 
"  Milk  ").  The  ideal  method,  and  one  which  commends  itself  at 
first  sight  as  being  the  most  satisfactory,  is  the  elimination  by 
slaughter  of  all  animals  which  are  tuberculous.  This  was  adopted 
in  the  State  of  Massachusetts  ;  under  an  order  of  the  Board  of 
Cattle  Commissioners  all  beasts  in  the  State  were  tested  with 
tuberculin,  and  every  animal  that  reacted  was  slaughtered,  and 
strict  quarantine  combined  with  the  tuberculin  test  imposed  on  all 
imported  cattle.  Even  in  this  small  State  such  a  plan  was  found 
to  be  unworkable,  the  expense  of  compensation  becoming  formid- 
able. A  middle  course  seems  to  be  the  only  practicable  one,  viz. 
all  manifestly  tuberculous  animals,  especially  where  wasting  or  a 
tuberculous  udder  is  present,  to  be  slaughtered  ;  other  animals  to 
be  tested  with  tuberculin,  and  those  which  react  to  be  separated 
from  the  healthy  and  to  be  disposed  of  (foi  slaughter)  as  soon  as 
convenient,  and  in  the  meanwhile  kept  as  much  as  possible  in  pasture. 

Avian  tubercle  bacilli  have  occasionally  been  met  with  in  man. x 

Tuberculosis  is  diminishing  among  the  white  races  ;  it  is,  how- 
ever, spreading  among  many  coloured  races.  It  is  to  be  noted  that 
the  decline  began  long  before  the  germ  origin  had  been  demonstrated, 
and,  what  is  more,  the  rate  of  decline  was  almost  as  great  before 
any  administrative  measures  were  taken  against  it  as  since.  Never  - 

1  Lowenstein,  Wien.  Klin.  Woch.,  May  15,  1913. 

21 


322  A  MANUAL  OF  BACTERIOLOGY 

theless,  it  can  hardly  be  doubted  that  measures  should  be  adopted 
by  local  authorities  and  others  to  prevent  the  spread  of  tuberculosis. 
All  forms  of  tuberculosis  have  now  been  made  notifiable  in  this 
country.  Patients  should  be  warned  of  the  danger  of  disseminating 
their  expectoration,  and  should  use  pocket-spittoons  containing  an 
antiseptic,  or  handkerchiefs  (such  as  the  Japanese  paper  ones) 
which  can  be  destroyed.  Rooms  which  have  been  inhabited  by 
tuberculous  patients  should  be  disinfected,  for  which  purpose 
Delepine  recommended  spraying  with  a  1-100  solution  of  chloride 
of  lime.  Although  the  occurrence  of  direct  infection  can  rarely  be 
proved,  the  possibility  of  this  cannot  be  ignored.  Not  only  should 
the  dissemination  of  infection  be  prevented,  but  the  resistance  of 
the  individual  should  be  raised  by  providing  a  healthy  environment 
and  by  inculcating  the  importance  of  fresh  air. 

Serum  therapeutics  and  vaccine. — Many  sera  have  been 
introduced  for  the  treatment  of  tuberculosis,  e.g.  Marag- 
liano's,  Marmorek's,  Spengler's,  Mehnarto's,  etc.  Speng- 
ler's  I.K.  serum  is  of  considerable  value  in  many  cases  : 
it  is  prepared  by  immunising  rabbits  by  intramuscular 
injections  and  contains  the  laked  red- corpuscles  as  well 
as  the  serum.1  Mehnarto's  is  stated  to  be  a  mixture  of 
sheep  and  snake  serums  and  is  reported  favourably  on  by 
Barcroft.2 

For  vaccine  treatment,  tuberculins  R  and  BE  are  usually 
employed  (p.  306).  Latham  has  found  that  tuberculin 
given  per  os  produces  its  characteristic  effects. 

Immunity. — Attempts  have  been  made  from  time  to 
time  to  produce  immunity  against  the  B.  tuberculosis, 
particularly  in  cattle.  Thus  McFadyean 3  found  that 
heifers  which  had  previously  been  subjected  to  repeated 
doses  of  tuberculin  (old)  in  some  cases  resisted  infection 
with  virulent  bacilli.  Behring  4  also  employed  human 
tubercle  bacilli  for  the  vaccination  of  cattle  with  satis- 

1  See  Treatment  of  Tuberculosis  by  Immune  Substances  (I.K.)  Therapy. 
Fearis  (John  Murray,  1912). 

2  British  Journ.  of  Tuberculosis,  1913. 

3  Trans.  Path.  Soc.  Lond.,  vol.  liii,  1902,  p.  20. 

4  Brit.  Med.  Journ.,  1906,  vol.  ii,  p.  577. 


COMPLEMENT  FIXATION  IN  TUBERCULOSIS  323 

factory   results.     His    tulase    likewise    confers   immunity 
when  given  either  by  the  mouth  or  by  the  stomach. 

Theobald  Smith x  also  concludes  that  vaccination  of 
calves  with  the  human  type  of  bacillus  is  harmless,  and 
that  the  procedure  leads  to  a  relatively  high  resistance  to 
fatal  doses  of  the  bovine  bacillus. 


Clinical  Examination 

I.  The  "  complement-fixation  "  test  was  first  used  in  tuberculosis 
by  Wassermann  and  Briick.  The  method  has  been  further  elaborated 
by  Emery.2  He  makes  use  of  a  standard  emulsion  of  tubercle 
bacilli  in  salt  solution,  containing  about  4  per  cent,  by  volume  of 
solid  bacillary  substance.  This  is  sterilised  by  intermittent  sterilisa- 
tion and  keeps  for  four  to  six  weeks.  Bacilli  from  various  sources 
vary  somewhat,  so  that  the  emulsion  should  be  standardised  so  as 
to  give  an  absorption-time  with  normal  sera  of  about  20  minutes, 
i.e.  the  complement  of  normal  serum  should  be  just  completely 
absorbed  in  about  20  minutes.  A  water-bath  kept  at  a  constant 
temperature  of  38°  C.  is  used  to  warm  all  the  constituents  and  mix- 
tures. One  part  of  the  serum  to  be  tested  is  mixed  with  four  parts 
of  the  bacillary  emulsion  in  a  small  tube  (e.g.  a  Durham's  tube)  in 
the  water-bath,  the  time  of  mixing  being  accurately  noted.  After 
2^  minutes'  incubation,  4  volumes  of  the  mixture  are  removed  by 
means  of  a  capillary  pipette  with  teat  (Fig.  35,  p.  215),  into  which 
also  a  single  volume  of  sensitised  corpuscles  (i.e.  a  hsemolytic 
system,  p.  184)  is  taken  up  and  the  whole  is  expelled  into  a  small 
tube  already  standing  in  the  water-bath.  The  process  is  repeated 
after  5,  10,  15,  and  20  minutes,  and  longer  if  necessary.  By  the 
occurrence  or  absence  of  haemolysis  in  the  various  tubes,  the  time 
taken  for  the  absorption  of  complement  is  ascertained,  the  comple- 
ment used  being  that  contained  in  the  serum  itself,  which  therefore 
should  be  fresh.  A  control  with  normal  serum  should  always  be 
performed  at  the  same  time.  With  normal  serum  complete  absorp- 
tion should  take  place  in  about  20  minutes  ;  with  tuberculous  sera 
it  is  often  complete  in  2|  minutes.  If,  then,  absorption  of  comple- 
ment is  complete  in  much  less  than  the  time  necessary  for  absorption 
with  a  normal  serum,  presumably  the  serum  is  derived  from  a 
tuberculous  individual.  (But  see  Emery's  paper  for  limitations.) 

1  Journ.  Med.  Research,  vol.  xviii,  1908,  p.  451. 

2  Lancet,  1911,  vol.  i,  p.  485. 


324  A  MANUAL  OF  BACTERIOLOGY 

II.  Precipitin    reaction. — Spengler    has     devised    a    precipitin 
reaction  for  the  diagnosis  of,  and  prognosis  in,  tuberculosis.     The 
reagents  are  the  blood-serum  or  the  laked  whole  blood,  or  both, 
very  highly  diluted  and  mixed  in  different  dilutions  with  tuber- 
culin.1 

III.  Agglutination  reaction. — The  method  of  agglutination  was 
proposed  by  Arloing  and  Courmont  for  the  diagnosis  of  tuber- 
culosis, but  is  difficult  to  carry  out  and  is  not  much  employed. 
A  special   method   has   to   be   employed  to   obtain   homogeneous 
cultures   of   the   tubercle   bacillus   or  a   powder  of  pulverised   or 
ground-up  bacilli  may  be  used  :    this  powder  may  be  purchased. 
The  reaction  may  be  carried  out  either  microscopically  or  macro- 
scopically  ;  for  the  latter  small  sterile  test-tubes  may  be  employed. 
For  each  test  three  dilutions  of  the  serum  are  made,  a  1  in  5,  a 
1  in  10,  and  a  1  in  20,  and  the  tubes  filled  with  these  dilutions  are 
allowed  to  stand  in  an  inclined  position  (45°)  for  five  to  ten  hours. 
In  man  the  serum  of  normal  individuals  may  agglutinate  up  to  a 
dilution  of  1  in  5,  while  in  animals  this  is  variable — imperceptible 
in  the  guinea-pig,  rabbit,  and  calf  ;  feeble  in  the  goat  ;  in  the  adult 
ox  up  to  1  in  5,  but  in  the  dog  it  may  be  up  to  1  in  10  or  even  1  in  20. 

A  positive  serum  reaction  in  a  suspected  subject  is  a  sign  of  great 
value  in  establishing  the  diagnosis  ;  a  negative  serum  reaction  is 
of  less  value. 

IV.  The  examination  of  sputum,  etc.,  for  the  tubercle  bacillus  is 
a  routine  procedure  of  the  greatest  value  in  forming  a  diagnosis. 
Fortunately,  owing  to  the  peculiar  staining  reaction  of  the  tubercle 
bacillus,  the  method  is  comparatively  simple. 

If  it  is  inconvenient  to  examine  the  sputum  for  a  day  or  two  a 
little  1-20  carbolic  should  be  added.  This  preserves  the  sputum 
and  the  tubercle  bacilli  retain  their  staining  power  for  some  time. 

1.  Sputum. — Film  specimens  are  prepared  by  smearing  a  little 
of  the  sputum  on  to  a  slide  with  a  needle  so  as  to  form  a  thin  film 
covering  two-thirds  of  the  surface,  or  by  placing  a  particle  of  the 
sputum  on  one  slide,  applying  another  slide,  pressing  together,  and 
then  drawing  apart  so  that  a  thin  film  is  left  on  each  slide.  The 
thick  portion  of  the  sputum  should  be  used,  the  thin  mucoid  portion 
being  rejected.  If  the  sputum  is  thin  and  watery,  the  thicker 
portion  can  be  obtained  by  covering  the  bottom  of  a  Petri  dish 
with  filter-paper,  placing  a  large  drop  of  the  sputum  on  this,  and 
working  it  over  the  paper  with  a  bent  steel  needle.  The  paper 
absorbs  the  water,  leaving  the  thicker  material  on  the  surface.  If 
there  are  any  small  yellow  caseous  particles  present  these  should 
1  See  Fearis,  Practitioner,  i,  1913. 


ZIEHL-NEELSEN  METHOD  325 

be  chosen,  and  sufficient  material  should  be  used  so  as  to  form  a 
distinct  but  not  too  thick  film  ;  a  little  experience  will  soon  decide 
the  right  amount  ;  too  thin  a  film  should  be  avoided.  Preparations 
may  also  be  made  by  smearing  the  sputum  on  a  cover-glass  or 
between  two  cover-glasses  instead  of  using  slides.  Whichever  plan 
is  adopted,  the  film  is  dried  and  fixed  in  the  usual  manner  (generally 
by  heat),  and  then  stained  by  one  of  the  following  methods  : 

(a)  Ziehl-N eelsen  mzthod. — Film  specimens  on  slides  are  most 
conveniently  stained  by  flooding  with  filtered,  undiluted  carbol- 
fuchsin  and  warming  for  2  to  5  minutes  on  a  piece  of  asbestos 
cardboard  supported  on  a  tripod,  or  on  a  heated  penny  (p.  110),  or 
slides  or  cover-glasses  flooded  with  the  stain  may  be  held  in  the 
forceps  and  carefully  warmed  over  a  flame,  or  the  preparations 
may  be  immersed  in  a  watch-glass  or  dish  of  the  stain,  covered,  and 
placed  in  the  warm  incubator  for  half  an  hour.  In  no  case  must 
the  stain  bs  allowed  to  boil,  or  the  bacilli  may  lose  their  staining 
power  ;  it  should  only  bs  warmed  sufficiently  to  steam  (50°-60°  C.), 
and  with  slides  or  cover-glasses  as  evaporation  takes  place  more 
stain  (always  filtered),  or  better,  5  per  cent,  carbolic,  should  be  added. 
After  staining,  the  preparations  are  rinsed  in  water  and  are  then 
decolorised  by  treating  with  25  per  cent,  sulphuric  or  30  per  cent, 
nitric  acid.  The  preparation  may  be  flooded  with  the  acid,  but  a 
better  method  is  to  immerse  the  preparation  in  a  pot  (Fig.  20, 
p.  110)  containing  the  acid.  In  the  acid  the  colour  changes  after 
a  few  seconds  to  a  yellowish  brown,  the  preparation  is  then  rinsed 
in  water,  and  some  of  the  pink  colour  returns.  The  treatment  with 
acid  and  with  water  alternately  is  repeated  until  the  preparation 
is  nearly  colourless  when  rinsed  in  water.  With  sputum  this  is 
usually  the  case  after  three  or  four  rinses  in  the  acid,  but  it  varies 
with  the  thickness  of  the  film  and  with  the  number  of  tubercle 
bacilli  present  ;  when  these  are  absent  the  film  often  decolorises 
more  readily  than  when  there  are  many.  The  presence  of  blood 
renders  the  decolorisation  difficult.  After  decolorising  and  washing, 
the  preparations  are  stained  for  one  minute  in  Loffler's  methylene 
blue,  washed  in  water,  and  mounted  in  water,  or,  better,  dried  and 
mounted  in  Canada -balsam  or  cedar  oil.  When  the  preparation  is 
made  on  the  slide,  after  washing  and  drying,  it  can  be  examined 
directly  without  a  cover-glass  with  the  oil-immersion  after  applying 
a  drop  of  cedar  oil,  unless  a  permanent  specimen  is  desired,  in  which 
case  it  should  be  mounted  in  Canada-balsam. 

The  tubercle  bacilli  appear  as  delicate  red  rods,  often  beaded  or 
segmented,  on  a  blue  background  composed  of  cells,  mucus,  and 
putrefactive  or  other  bacteria.  Occasionally  here  and  there  a  little 


326  A  MANUAL  OF  BACTERIOLOGY 

red  colour  may  be  present  in  addition  to  the  tubercle  bacilli.  Hair 
and  keratinised  material  generally,  such  as  horny  epithelium,  and 
red  blood-corpuscles,  retain  the  red  colour  after  the  foregoing 
treatment,  and  the  spores  of  bacteria  are  also  liable  to  retain  the 
red  somewhat  persistently.  These  exceptions  are  not,  however, 
likely  to  prove  a  source  of  error,  for  the  tubercle  bacilli  should  be 
recognised  not  only  by  their  red  colour,  but  also  by  their  charac- 
teristic size,  shape,  and  general  appearance.  It  is  conceivable  that 
acid-fast  bacilli  not  tubercle  might  be  present  in  sputum,  but  such 
an  event  is  a  very  unlikely  one.  For  the  microscopical  examination, 
a  ^-inch  with  good  illumination  is  sufficient  when  the  tubercle  bacilli 
are  present  in  any  number.  When  they  are  scanty  it  is  necessary 
to  use  a  yV'inch  oil-immersion,  and  this  is  the  better  lens  in  any 
case.  (See" Plate  IX,  b,  and  Plate  X,  a.) 

If  tubercle  bacilli  are  not  found,  other  specimens  should  be  pre- 
pared and  examined.  It  is  only  by  repeated  examinations  on  different 
occasions  that  the  negative  evidence,  the  absence  of  tubercle  bacilli, 
becomes  of  any  value. 

The  tubercle  bacillus  is  occasionally  not  acid-fast  ;  1  probably 
the  bacilli  in  such  cases  are  degenerate,  and,  like  all  degenerate 
bacteria,  fail  to  stain  well.  Spengler  claims  that  the  following 
method  will  stain  these  and  "splitter"  forms:  (1)  Stain  with 
warm  carbol-fuchsin  by  the  ordinary  method,  avoiding  overheating  ; 
(2)  pour  off  the  stain  without  washing  and  treat  with  picric  acid 
alcohol  (equal  parts  of  saturated  aqueous  picric  acid  and  absolute 
alcohol)  ;  (3)  after  3  seconds  rinse  with  60  per  cent,  alcohol ; 

(4)  treat  with  15  per  cent,  nitric  acid  until  yellow  (about  30  seconds)  ; 

(5)  rinse  again  with  60  per  cent,  alcohol ;    (6)  counter-stain  with 
the  picric  acid  alcohol  until  yellow  ;   (7)  wash  with  distilled  water. 
This  is  an  excellent  method,  and  thick  films  may  be  used.     In 
material  which  has  been  preserved  a  long  time,  e.g.  sputum  with 
carbolic,  or  tissue  in  spirit,  the  bacilli  may  be  much  less  acid-fast 
than  in  fresh  material. 

Various  methods  have  been  recommended  for  the  solution  of 
the  sputum  and  the  examination  of  the  sediment  of  the  bacilli. 
In  one  method  5  c.c.  of  sputum  are  mixed  with  50  c.c.  of  normal 
KOH  solution  ;  the  mixture  is  warmed  in  a  water-bath  to  60°-65°  C. 
until  the  sputum  is  dissolved  (about  3  hours)  ;  50  c.c.  of  cold  water 
are  next  added,  the  whole  is  well  shaken,  and  again  warmed  for 
^  hour.  Petroleum  ether,  2  c.c.,  is  next  added,  the  whole  is  well 
shaken,  and  is  then  kept  at  60°  C.  until  the  ether  has  separated. 
The  bacilli  will  be  concentrated  in  the  fluffy  layer  at  the  junction 
1  See  Lancet,  1908,  vol.  i,  p.  1222. 


MUCH'S  METHOD  327 

of  the  ether  and  water  ;  this  is  pipetted  off  and  films  are  made  with 
it  and  stained.  Antiformin  (a  mixture  of  sodium  hypochlorite  and 
sodium  hydrate)  has  also  been  recommended.  Into  a  boiling- tube 
or  small  flask  of  50  c.c.  capacity,  5  c.c.  of  the  sputum  are  introduced. 
To  this  are  added  25  c.c.  of  antiformin  solution  (10-20  per  cent, 
aqueous  solution)  diluted  with  10-20  c.c.  of  water  according  to  the 
density  of  the  sputum.  The  mixture  is  well  shaken  until  homo- 
geneous (about  15  minutes),  then  centrifuged,  the  deposit  is  washed 
three  times  with  salt  solution  by  centrifuging,  and  films  are  made 
with  the  washed  deposit  and  stained  by  the  Ziehl-Neelsen  or 
Spengler  method. 

If  the  tubercle  bacillus  cannot  be  detected  microscopically  after 
repeated  examinations,  and  a  certain  diagnosis  is  important,  the 
inoculation  method  may  be  employed.  A  couple  of  guinea-pigs  are 
inoculated  subcutaneously  in  the  thigh  or  abdomen  with  0-5  to  1  c.c. 
of  the  sputum.  If  tubercle  bacilli  are  present  the  animals  will 
show  signs  of  tuberculosis  in  three  to  six  weeks  (see  below, 
"  Urine  "). 

(6)  Other  methods  have  been  devised  for  staining  the  tubercle 
bacillus,  but  do  not  seem  to  be  better  than  the  Ziehl-Neelsen  or  the 
Spengler.  The  following  may  be  useful  for  those  who  are  colour- 
blind to  red : 

a.  Muck's  method. — Prepare  the  following  solution  :  10  c.c.  of 
a  saturated  alcoholic  solution  of  methyl  violet  B.N.  in  100  c.c.  of 
2  per  cent,  aqueous  carbolic  ;  (1)  stain  the  film  with  this,  warming 
over  the  flame,  or  for  24-48  hours  at  37°  C.  ;  (2)  treat  with  Gram's 
iodine  solution,  1-5  minutes  ;  (3)  treat  with  5  per  cent,  nitric  acid 
for  1  minute  ;  (4)  treat  with  3  per  cent,  hydrochloric  acid  for 
10  seconds  ;  (5)  treat  with  a  mixture  of  equal  parts  of  acetone  and 
absolute  alcohol. 

/3.  Herman's  method. — Prepare  shortly  before  use  the  following 
solution  :  3  parts  of  a  1  per  cent,  aqueous  solution  of  ammonium 
carbonate,  1  part  of  a  3  per  cent,  solution  of  krystal  violet  in  95  per 
cent,  methyl  alcohol.  (1)  Flood  the  film  with  this,  warm  until  it 
steams,  and  stain  for  1  minute  ;  (2)  decolorise  with  10  per  cent, 
nitric  acid  for  a  few  seconds,  and  then  with  95  per  cent,  alcohol 
until  the  film  assumes  a  pale  blue  colour,  then  rinse  in  tap-water 
followed  by  distilled  water  ;  (3)  counter-stain  with  1  per  cent, 
aqueous  eosin. 

By  both  these  methods  the  tubercle  bacilli  appear  blue-black. 
2.  Tissues. — The  histological  appearance  of  the  tubercle  is  usually 
sufficient  for  diagnostic  purposes  without  the  demonstration  of  the 
tubercle  bacilli,  which  in  many  instances  may  be  difficult  in  human 


328  A  MANUAL  OF  BACTERIOLOGY 

material,  as  the  bacilli  may  be  very  scanty,  or  practically  impossible 
to  find,  e.g.  in  lupus.  Sections  should  be  prepared  either  by  the 
freezing  or  the  paraffin  method,  stained  with  haematoxylin,  and 
counter-stained  with  eosin,  or  orange-rubin,  or  with  the  Ehrlich- 
Biondi  mixture. 

In  order  to  demonstrate  the  tubercle  bacillus  in  fresh  tissue 
smears  may  be  made  and  stained  like  sputum,  or  sections  prepared 
and  stained  in  warm  carbol-fuchsin  for  about  ten  minutes.  For 
frozen  sections  the  stain  may  be  contained  in  a  watch-glass  or  small 
glass  capsule,  and  is  warmed  until  it  steams,  but  not  boiled,  on  a 
piece  of  asbestos  cardboard  or  a  sand-bath.  Paraffin  sections 
should  be  fixed  to  the  slides  with  glycerin  albumin,  and  may  be 
stained  by  flooding  with  the  carbol-fuchsin  and  warming  on  asbestos 
cardboard,  or  a  heated  penny,  for  ten  minutes.  After  staining,  the 
sections  are  washed  in  water  and  are  then  decolorised  in  25  per 
cent,  sulphuric  acid.  This  is  a  longer  process  than  with  sputum, 
and  the  sections  after  being  in  the  acid  for  a  few  seconds  are  washed 
in  water  and  then  returned  to  the  acid,  and  this  alternate  rinsing  in 
acid  and  in  water  is  repeated  until  they  are  nearly  colourless  when 
placed  in  water.  It  is  not  necessary  to  remove  the  colour  absolutely  ; 
a  faint  pink  remaining  does  not  matter.  After  rinsing  in  fresh  water 
to  remove  all  the  acid,  the  sections  are  counter-stained  in  Loffler's 
methylene  blue  for  two  minutes,  rinsed  in  methylated  spirit,  passed 
through  absolute  alcohol  somewhat  rapidly  to  avoid  removing  too 
much  of  the  blue,  cleared  in  cedar  oil  or  xylol,  and  mounted  in 
balsam.  The  sections  may  also  be  counter-stained  with  haema- 
toxylin or  Bismarck  brown. 

Instead  of  using  the  strong  acid  solution  for  decolorising,  an 
acid  alcohol  solution  may  be  used  with  advantage,  or  2  per  cent, 
aqueous  hydrochloride  of  anilin  may  be  employed. 

Gram's  method  may  also  be  used,  but  is,  of  course,  not  distinctive 
for  the  tubercle  bacillus. 

Sections  may  also  be  first  stained  with  Ehrlich's  or  other  haema- 
toxylin solution,  then  stained  with  warm  carbol-fuchsin,  washed, 
treated  with  2  per  cent,  aqueous  anilin  hydrochloride  for  a  few 
seconds,  decolorised  with  75  per  cent,  alcohol  until  the  red  colour 
is  no  longer  apparent  (15-30  minutes),  and  counter-stained  with  an 
aqueous  solution  of  orange. 

Where  a  positive  diagnosis  is  important,  a  small  piece  of  the 
tissue  may  be  inserted  under  the  skin  of  the  thigh  or  abdomen  of 
a  guinea-pig.  If  tuberculous,  the  animal  will  show  signs  of  tuber- 
culosis in  two  or  three  weeks  (see  below,  "  Urine  "). 

Films  of  pure  cultivations  of  the  tubercle  bacillus  may  be  stained 


TUBERCLE  BACILLUS  IN  URINE  329 

in  warm  carbol-fuchsin  for  two  to  five  minutes,  rinsed  in  the 
sulphuric  or  nitric  acid  solution,  washed,  dried,  and  mounted.  They 
can  also  be  stained  by  Gram's  method,  which  usually  brings  out 
the  beaded  appearance  very  markedly,  or  by  any  of  the  other 
methods  mentioned  under  Sputum.  Differentiation  from  the 
leprosy  bacillus  will  be  found  at  p.  337,  and  from  the  smegma 
bacillus  and  other  acid-fast  organisms  at  p.  339. 

3.  Urine. — The  tubercle  bacillus  is  often  very  difficult  to 
demonstrate  in  urine.  The  urine  must  be  allowed  to  stand  in  a 
conical  glass  for  twenty-four  hours  or  centrifuged,  and  film 
specimens  are  prepared  with  the  sediment  and  treated  by  one  of 
the  methods  for  sputum  given  above.  Several  specimens  should  be 
made  and  must  be  very  carefully  examined.  The  sediment  may 
also  be  treated  by  the  antiformin  method.  It  is  important  to 
exclude  the  smegma  bacillus,  and  the  urine  is  preferably  drawn  off 
by  a  catheter.  Staining  may  be  carried  out  by  Housell's  method, 
by  which  the  smegma  bacillus  is  decolorised,  viz.  after  staining  in 
warm  carbol-fuchsin  the  specimen  is  washed  and  dried.  It  is  then 
immersed  in  acid  alcohol  (alcohol  +  3  per  cent,  hydrochloric)  for 
ten  minutes,  washed  in  water,  counter-stained  for  a  few  seconds  in 
a  saturated  alcoholic  solution  of  methylene  blue,  washed,  dried, 
and  mounted  (see  also  p.  339).  An  electrolytic  method  for  the 
concentration  of  the  tubercle  bacilli  has  been  devised  by  Russ.1 

If  a  diagnosis  is  of  importance  inoculation  should  be  resorted  to. 
Two  guinea-pigs  are  inoculated  subcutaneously  in  the  thigh  or 
abdomen  with  0-5  to  1  c.c.  of  the  deposit  from  the  sedimented  or 
centrifuged  urine,  or  one  may  be  inoculated  subcutaneously,  the 
other  intra-peritoneally.  If  tubercle  bacilli  are  present  the  animals 
may  show  signs  of  tuberculosis  as  early  as  two  to  three  weeks  after 
inoculation.  Sometimes,  of  course,  the  animals  may  die  from  some 
intercurrent  infection  before  the  tuberculous  infection  has  had  time 
to  develop  Delepine  2  recommends  the  inoculations  to  be  made 
on  the  inner  aspect  of  the  leg  about  the  level  of  the  knee.  The 
order  of  infection  after  inoculation  is  as  follows  :  the  popliteal, 
superficial  and  deep  inguinal,  and  sub-lumbar  glands,  the  retro- 
hepatic,  mediastinal  and  bronchial,  deep  servical,  and  subscapular 
glands,  the  spleen,  liver,  and  lungs.  The  inoculated  animals  are 
killed  in  two  to  three  weeks,  dissected,  and  the  lesions  examined 
microscopically.  Others  inoculate  two  guinea-pigs,  one  sub- 

1  Proc.  Roy.  Soc.  Lond.,  B.  1909. 

2  Brit.  Med.  Journ.,  1893,  vol.  ii,  p.  664.     The  results  only  apply  to 
ordinary  forms  of  tuberculosis,  and  not  to  certain  modified  forms  such 
as  lupus  and  the  avian  variety. 


330  A  MANUAL  OF  BACTERIOLOGY 

cutaneously  in  the  abdomen,  the  other  intra-peritoneally.  Negative 
results  are  nearly  as  valuable  as  positive  ones. 

In  fceces,  if  definite  yellow  caseous  particles  can  be  found,  these 
should  be  picked  out,  and  films  made  and  stained.  Antiformin 
may  also  be  used.  About  5-6  c.c.  of  faeces  are  mixed  with  20  c.c. 
of  15  per  cent,  aqueous  antiformin  in  a  conical  glass,  well  agitated 
and  broken  up,  and  an  equal  volume  of  the  dilute  antiformin  is 
then  added.  The  mixture  is  allowed  to  stand  for  an  hour,  and  films 
are  prepared  from  the  white  curdy  layer  which  forms,  stained,  and 
examined. 

4.  M ilk. — See  section  on  milk  (Chapter  XXI). 

V.  The  opsonic  method. — The  general  mode  of  carrying  this  out 
is  described  at  pp.  314-319,  the  tubercle  bacilli  being  suspended  in 
1-5  per  cent,  salt  solution. 

VI.  Tuberculin  reactions. — The  old  tuberculin  is  used  for  diagnostic 
purposes  ;  it  is  not  perhaps  very  safe.     A  dose  of  0-0002  c.c.  is 
injected  subcutaneously,  and  the  temperature  taken  four-hourly 
during  the  succeeding  thirty-six  hours.     A  rise  of  2°-3°  F.  or  more 
ensues  a  few  hours  after  injection  in  tuberculous  subjects.     If  no 
reaction  occurs  another  dose  of  0-0005  c.c.  may  be  given  after  the 
lapse  of  some  days. 

This  method  has  now  almost  completely  been  superseded  by  the 
cutaneous  or  by  the  ophthalmo  reaction. 

The  cutaneous  tuberculin  reaction. — Von  Pirquet  1  discovered  that 
when  tuberculin  is  introduced  into  the  superficial  layers  of  the  skin 
of  tuberculous  individuals,  as  in  vaccination,  a  reaction  occurs 
consisting  of  the  formation  of  a  papule  with  redness,  slight  swelling 
and  exudation,  and  sometimes  small  vesicles.  This  reaction  is 
usually  at  its  height  twenty-four  to  forty-eight  hours  after  inocula- 
tion. In  healthy  individuals  no  reaction  follows  the  inoculation. 
The  method  is  to  scarify  a  small  spot  on  the  forearm  through  a  drop 
of  a  dilution  of  the  old  tuberculin,  and  protect  the  patch  with  a 
simple  dry  dressing.  Moro  has  modified  the  method  by  applying 
the  tuberculin  to  the  skin  in  the  form  of  ointment. 

The  ophthalmo-tuberculin  reaction. — Calmette  transferred  the  site 
of  inoculation  from  the  skin  to  the  conjunctiva.  He  makes  use  of 
material  prepared  by  precipitating  the  old  tuberculin  with  alcohol, 
of  which  a  1-100  solution  is  prepared  in  distilled  water.  One  drop 
of  this  is  instilled  into  the  inner  half  of  the  conjunctiva  of  one  eye. 
In  tuberculous  individuals  a  reaction  follows,  usually  in  six  to  sixteen 
hours  after  medication,  consisting  of  a  conjunctivitis,  ranging  in 
intensity  from  a  local  redness  to  a  redness  extending  over  the  whole 
1  Wien.  msd.  Woch.,  July  6,  1907. 


PSEUDO-TUBERCULOSIS  331 

eye  and  having  the  appearance  of  an  acute  conjunctivitis.  The 
reaction  soon  passes  off,  generally  without  leaving  ill  effect.  On 
the  whole  the  reaction  appears  to  be  fairly  constant  in  tuberculous 
individuals,  but  absence  of  reaction  is  not  certain  proof  that  the  case 
is  not  tuberculous.1 

VII.  Tuberculin  for  veterinary  use. — The  dose  of  the  various 
preparations  in  the  market  varies  according  to  their  strength  ;  it 
corresponds  to  0-1  c.c.  or  0-2  c.c.  of  Koch's  original  tuberculin. 

The  appropriate  dose  is  injected  subcutaneously  in  the  neck  and 
the  reaction  consists  of  a  rise  of  temperature  of  from  1-5°  to  6°  F. 
above  the  average  normal,  commencing  8-12  hours  after  injection 
and  lasting  12-14  hours,  the  temperature  being  taken  at  the 
twentieth  hour  after  injection,  or,  if  it  can  be  done,  at  frequent 
intervals  from  the  twelfth  to  the  twentieth  hour.  The  temperature 
should  be  taken  just  before  inoculation,  and,  if  possible,  morning 
and  evening  for  two  or  three  days  previous  to  inoculation. 

A  healthy  animal  is  unaffected  by  the  injection,  and  if  an  animal 
be  extensively  affected  with  tuberculosis  the  reaction  may  not  be 
given,  or  may  be  masked  by  the  fever  present. 

An  ophthalmo-reaction  may  also  be  employed  in  cattle. 

Johne's  disease,2  a  bovine  enteritis,  is  due  to  an  acid-fast  bacillus 
closely  resembling  the  tubercle  bacillus  in  morphology.  It  occurs 
in  scrapings  of  the  affected  mucous  membrane  of  the  bowel,  and  also 
in  sections  of  the  intestinal  wall.  The  Johne  bacillus  is  inoculable 
into  the  goat,  but  not  into  the  guinea-pig  or  rabbit,  and  does  not 
grow  on  any  of  the  ordinary  laboratory  media.  Twort  states  that 
it  can  be  cultivated  on  the  medium  employed  by  him  for  growing 
the  leprosy  bacillus  (p.  335,  and  from  the  cultures  a  diagnostic) 
vaccine  may  be  prepared.3 


Pseudo-Tuberculosis 

The  term  "  pseudo- tuberculosis  "  (which  is  not  a  good 
one,  and  should  be  discarded)  has  been  applied  to  a  number 
of  different  conditions  which  have  as  a  common  character 
the  presence  of  tubercle-like  nodules,  but  which  are  not 
caused  by  the  tubercle  bacillus.  Such  are  produced  by 

1  See  Brit.  Med.  Journ.  and  Lancet,  1907,  vol.  ii,  and  1908,  vol.  i. 

2  See  MacFadyean,  Journ.  Comp.  Path,  and  Therap.,  vol.  xx,  1907, 
p.  48. 

3  Twort,  Veterinary  Record,  Sept.  14,  1912. 


332  A  MANUAL  OF  BACTERIOLOGY 

certain  parasitic  worms,  by  Blastomycetes,  Streptothrix  and 
Aspergillus,  Protozoa,  and  by  several  bacteria. 

PfeifFer's  Bacillus  pseudo-tuberculosis  produces  nodular 
deposits  in  the  organ,  accompanied  by  wasting,  very  like 
true  tuberculosis.  The  disease,  however,  runs  a  more 
rapid  course,  death  ensuing  in  the  guinea-pigs  two  to  three 
weeks  after  inoculation.  Guinea-pigs,  rabbits,  mice  and 
monkeys  can  be  readily  infected.  The  nodules  consist  of 
masses  of  round  cells  which  undergo  necrosis  and  caseation. 
The  bacillus  in  the  tissues  is  not  readily  stained,  carbol- 
methylene  blue  being  the  best  solution,  as  it  is  not  acid- 
fast,  nor  does  it  stain  by  Gram's  method.  Morpho- 
logically it  is  a  small  rod  1-2  /m  in  length,  usually  non- 
motile,  although,  according  to  Klein,  it  possesses  a  single 
flagellum  or  two  flagella  at  one  end.  On  gelatin  it  forms 
a  whitish  growth  without  liquefaction,  like  that  of  the 
colon  bacillus,  but  confined  to  the  needle-track.  It  pro- 
duces alkali,  forms  no  gas,  and  does  not  curdle  milk. 
Broth  remains  clear,  with  a  whitish  stringy  flocculent 
deposit.  The  bacillus  grows  readily  and  rapidly. 

MacConkey  has  found  that  the  fermentation  reactions 
of  this  organism  and  of  the  plague  bacillus  are  practically 
identical  (see  "  Plague,"  p.  395),  and  sterilised  cultures  of 
either  will  protect  against  the  other. 

Ovine  caseous  lymphadenitis,  a  disease  of  sheep  simu- 
lating tuberculosis,  is  due  to  a  short  pump  bacillus  with 
rounded  ends  which  stains  well  by  Gram's  method,  and 
grows  best  on  blood-serum,  on  which  it  forms  greyish 
colonies.1 

Much  finds  in  the  glands  in  Hodgkin's  disease  anti- 
formin- resistant  bodies,  non-acid-fast,  and  similar  to  the 
non-acid-fast  tubercle  bacilli  which  he  has  described. 

1  Sixteenth  Ann.  Rep.  Bureau  of  Animal  Indust.  U.S.A.,  p.  638. 


LEPROSY  333 

Leprosy 

Leprosy,  elephantiasis  Graecorum  or  true  elephantiasis 
is  a  disease  of  which  we  have  records  from  the  earliest 
times.  It  was  undoubtedly  somewhat  prevalent  in  the 
British  Isles  from  the  twelfth  to  the  fifteenth  centuries, 
as  the  many  leper  houses  and  enactments  against  lepers 
testify,  though  no  doubt  other  skin  diseases,  psoriasis, 
lupus,  etc.,  were  at  that  early  period  of  medical  diagnosis 
confounded  with  it.  At  the  present  day  leprosy,  although 
extinct  in  the  British  Isles,  may  be  said  to  have  a  world- 
wide distribution,  for  it  is  met  with  in  Iceland  and  Scan- 
dinavia, Russia  and  the  Mediterranean  coasts  ;  in  Persia, 
India,  China,  Siberia,  and  Japan  ;  in  Africa  from  north  to 
south  ;  in  many  districts  of  the  American  continent ;  and 
in  the  Pacific  Islands.  Three  varieties  of  leprosy  are 
described — the  tuberculated  or  nodular,  the  anaesthetic, 
and  the  mixed. 

The  mode  of  spread  is  probably  by  personal  contact 
(though  possibly  insects  play  some  part),  and  throughout 
ancient  and  mediaeval  times  leprosy  was  considered  to 
be  a  contagious  and  communicable  disease,  as  witness  the 
stringent  regulations  in  the  Mosaic  and  other  laws  for  the 
segregation  of  lepers.  J.  Hutchinson  supposed  that  fish 
in  the  diet,  particularly  if  stale,  decomposed,  or  badly 
cured,  in  some  way  is  a  causative  factor ;  but  he  is 
practically  alone  in  this  view. 

A  bacillus,  the  Bacillus  leprce,  is  abundant  in  the  tissues 
and  was  discovered  by  Hansen  in  1879.  In  form  it 
resembles  the  tubercle  bacillus,  but  is  slightly  more  slender  ; 
it  probably  does  not  form  spores,  though  in  stained  pre- 
parations the  same  irregularity  in  staining — namely,  the 
occurrence  of  unstained  intervals,  the  so-called  "  beading  *' 
— is  met  with  as  in  the  tubercle  bacillus,  and  is  assumed 
by  some  to  be  due  to  the  presence  of  spores.  The  organism 


334  A  MANUAL  OF  BACTERIOLOGY 

as  obtained  from  the  tissues  is  non-motile,  stains  readily 
with  the  ordinary  anilin  dyes,  and  by  Gram's  method, 
which  brings  out  the  beaded  appearance  very  well,  and  is 
markedly  acid-fast,  thus  closely  resembling  the  tubercle 
bacillus,  and  the  methods  used  to  demonstrate  it  are  the 
same  as  for  the  latter  organism. 

The  Bacillus  leprce  is  found  in  enormous  numbers, 
usually  crowded  together  in  bundles  or  masses,  in  the 
leprous  nodules  in  the  skin  (Plate  X.  a),  liver,  spleen,  and 
testicles,  in  the  affected  nerves  in  the  anaesthetic  form 
and  even  in  the  ganglion  cells  of  the  central  nervous  system 
— in  fact,  any  viscus  may  be  affected  ;  it  has  also  been 
found  in  the  blood,  but  only  in  the  febrile  paroxysms 
which  set  in  when  the  disease  is  approaching  a  fatal 
termination.  The  exact  situation  of  the  leprosy  bacilli 
in  the  tissues  has  been  a  matter  of  controversy.  By  some 
it  has  been  held  that  they  are  contained  within  certain 
round  cells,  the  so-called  leprous  cells,  and  this  may  be 
the  case,  but  to  an  inconsiderable  extent.  Unna  has 
always  regarded  these  leprous  cells  as  really  being  trans- 
verse sections  of  lymphatic  vessels  containing  bacillary 
thrombi,  and  this  seems  to  be  usually  the  case.  Giant- 
cells  are  occasionally  present  in  the  leprous  nodules.  One 
of  the  most  constant  and  earliest  situations  in  which  the 
B.  leprce  is  found  is  the  nasal  mucous  membrane. 

Lepers  react  to  the  old  tuberculin  and  also  give  the 
Wassermann  reaction. 

Although  the  organism  is  present  in  such  enormous 
numbers  and  is  so  readily  demonstrable,  to  cultivate  it 
on  artificial  media  and  to  infect  animals  with  it  are  both 
difficult  matters.  Babes,  Bordoni-Uffreduzzi,  Czaplewski, 
are  some  of  those  who  in  the  past  believe  that  they  have 
cultivated  the  leprosy  bacillus.  Van  Houten1  claimed  to 
have  succeeded  by  growing  it  in  glycerin  fish  broth.  The 

1  Journ.  Path,  and  BacL,  vol.  viii,  1903,  p.  260. 


LEPROSY  BACILLUS  335 

bacillus  cultivated  was  acid-fast,  and  agglutinated  with, 
and  was  sensitised  by,  lepers'  serum. 

Deycke,1  by  taking  fragments  of  leprosy  tissue  and 
incubating  for  several  weeks  in  physiological  salt  solution 
at  37°  C.,  obtained  a  growth  of  a  semi-acid-fast  strepto- 
thrix,  S.  kproides.  He  is  uncertain  if  this  is  a  true  growth 
of  the  leprosy  bacillus.  Injected  into  leprosy  patients  it 
seemed  to  produce  a  beneficial  effect.  The  acid-fast 
property  resides  in  a  fatty  substance  which  can  be  extracted 
with  solvents,  particularly  benzoyl  chloride.  The  fatty 
substance  Deycke  terms  "  nastin  "  ;  it  is  a  neutral  fat, 
the  glycerin  ester  of  a  fatty  acid  of  high  molecular  weight. 
Injected  into  leprosy  patients  it  sometimes  produces 
marked  reaction,  sometimes  not.  In  solution  in  benzoyl 
chloride  it  is  much  more  active,  and  Deycke  hopes  that  it 
will  act  as  a  curative  vaccine  in  leprosy.  On  the  whole, 
the  results  obtained  with  nastin  have  been  disappointing. 
Twort2  claimed  to  have  cultivated  the  B.  leprce  on  a 
medium  consisting  of  eggs,  glycerin,  and  ground-up 
tubercle  bacilli.  Clegg  states  that  the  leprosy  bacillus 
will  grow  in  symbiosis  with  amoebae,  and  Duval  that  it 
grows  in  1  per  cent,  human  serum  in  symbiosis  with  some 
bacteria.  Kedrowsky  and  Bayon  claim  to  have  grown  the 
organism  on  a  placental-juice  agar,  and  Bayon  has  obtained 
complement  fixation  with  his  cultures  with  leper  serum. 
Kedrowsky's  organism  is  a  non-acid-fast  diphtheroid, 
Clegg's  an  acid-fast  chromogenic  bacillus,  Duval's  and 
Bayon's  are  acid-fast  leproid  bacilli. 

In  1904  Rost  announced  that  he  had  obtained  cultures 
of  the  leprosy  bacillus  in  a  chlorine-free  medium,  but  this 
was  not  confirmed.  In  1909  he  again  claimed  success  by 
cultivating  in  a  medium  consisting  of  the  fluid  obtained 
by  the  steam  distillation  of  rotten  fish  to  which  is  added 

1  Brit.  Med.  Journ.,  1908,  vol.  i,  p.  802. 

2  Proc.  Roy.  Soc.  Lond.,  B.,  1911. 


336  A  MANUAL  OF  BACTERIOLOGY 

a  little  Lemco  broth  and  milk,  and  Bannerman  believes 
that  he  is  correct.1  Williams  has  grown  a  non-acid-fast 
streptothrix  in  ordinary  broth,  and  has  also  cultivated 
acid- fast  bacilli  in  a  modified  Rost  medium  (substituting 
distilled  water  for  the  fish  distillate).  The  writer  has  also 
grown  a  non- acid- fast  streptothrix  from  a  case  of  leprosy 
on  brain  agar  containing  the  juice  from  disintegrated 
B.  megaterium.  As  a  result  of  these  alleged  positive 
cultural  results,  it  has  been  surmised  that  the  B.  leprce  is 
really  a  streptothrix,  that  it  is  acid- fast  only  under  certain 
conditions,  viz.  in  the  body  or  in  media  containing  fat, 
and  that  under  cultivation  the  streptothrix  may  break  up 
into  non-acid-fast  diphtheroid  bacilli  or  into  acid-fast 
leproid  bacilli.  On  the  other  hand,  Fraser  and  Fletcher2 
have  made  373  inoculations  from  33  non- ulcerating  cases 
of  leprosy  on  a  variety  of  culture  media  with  entirely 
negative  results.  More  work  is  therefore  required  before 
it  can  be  definitely  stated  that  the  leprosy  bacillus  has 
been  cultivated. 

A  certain  number  of  positive  results  of  the  inoculation 
of  leprous  material  into  the  lower  animals  have  been 
reported  by  Ortmann  and  others.  Nicolle3  has  reported 
the  successful  inoculation  of  a  macaque  monkey,  but 
most  of  the  attempts  have  ended  in  failure ;  positive 
results  are  open  to  criticism  and  may  be  fallacious,  for 
lepers  not  infrequently  suffer  from  coincident  tuberculosis, 
and  the  animals  therefore  may  have  been  infected  with 
tuberculosis.  Japanese  dancing  mice  are  also  stated  to  be 
slightly  susceptible.  The  local  lesion  induced  in  animals 
may  be  simply  inflammatory,  produced  by  the  leprous 
material  acting  as  a  foreign  body,  and  the  bacilli  may  be 
diffused  without  proliferating.  Human  beings  have  also 

1  See  Sc.  Mem.  Gov.  of  India,  No.  42,  1911. 

2  Lancet,  Sept.  27,  1913. 

3  Comp.  Rend.  Acad.  Sc.,  1905. 


DIAGNOSIS  OF  LEPROSY  337 

been  inoculated,  but  the  positive  results  obtained  are  all 
open  to  objection. 

The  differentiation  of  leprosy  from  tuberculosis,  although 
the  bacilli  are  so  similar,  does  not  in  the  majority  of  cases 
present  much  difficulty.  The  large  number  of  bacilli 
present  in  the  lesions,  and  particularly  in  the  skin,  forms 
a  marked  distinction  from  tuberculosis.  The  Bacillus 
leprce  also  stains  more  readily,  and  with  watery  solutions 
in  a  shorter  time,  than  does  the  Bacillus  tuberculosis, 
though  this  distinction  is  hardly  marked  enough  for 
diagnostic  purposes. 

Cases  of  leprosy,  both  of  the  nodular  and  anesthetic 
varieties,  have  been  treated  with  injections  of  Koch's 
tuberculin,  which  has  been  found  to  produce  a  certain 
amount  of  reaction  followed  by  some  amelioration  in 
their  condition.  Rost  and  Williams  with  their  cultures 
have  prepared  vaccines  with  which  treatment  is  being  pur- 
sued. Nicholls  and  others  have  used  extracts  of  leprous 
tissue  as  a  vaccine,  and  Bayon  states  that  a  filtered  extract 
of  the  Kedrowsky  culture  is  of  service  for  treatment. 

Dean  x  and  others  have  met  with  a  leprosy-like  disease  in  the  rat. 
Marchoux  found  about  5  per  cent,  of  the  sewer  rats  in  Paris  infected 
with  it.  Nodules  are  found  in  the  tissues  which  contain  large 
numbers  of  an  acid-fast  bacillus  closely  resembling  the  B.  leprce^ 
Material  from  infected  rats  inoculated  into  healthy  rats  reproduces 
the  disease  after  some  months,  but  has  no  effects  on  guinea-pigs. 
The  disease  is  probably  conveyed  by  contact. 

Dean  cultivated  a  diphtheroid  non-acid-fast  bacillus  from  this 
disease  ;  Bayon  an  acid-fast  leproid  bacillus  which  he  finds  to  be 
very  similar  to  that  obtained  by  him  from  human  leprosy. 

Clinical  Examination 

(1)  If  cutaneous  nodules  be  present,  one  is  clamped,  pricked,  and 
films  are  prepared  with  the  juice  that  exudes  and  stained  as  for 

1  Journ.  of  Hyg.,  vol.  v,  1905,  p.  99  ;  Marchoux  and  Sorel,  Ann.  de 
Vlnst.  Pasteur,  xxvi,  1912,  p.  778. 

22 


338  A  MANUAL  OF  BACTERIOLOGY 

tubercle.  The  occurrence  of  large  numbers  of  bacilli,  having  the 
same  staining  reactions  as  the  tubercle  bacillus  and  obtained  from 
the  cutaneous  structures,  is  diagnostic  of  leprosy  (the  smegma 
bacillus  may  be  present  on,  but  not  in,  the  skin). 

(2)  In  the  tissues,  sections  of  which  are  stained  in  the  same 
manner  as  tuberculous  material,  the  diagnosis  must  be  based  on  the 
presence  of  the  bacilli  in  large  numbers  in  the  so-called  leprosy -cells. 

(3)  Leprosy  is  not  inoculable  in  guinea-pigs. 

N.B. — It  must  be  remembered  that  lepers  not  infrequently  suffer 
from  coincident  tuberculosis. 

(4)  The  differentiation  of  the  leprosy  from  the  tubercle  bacillus 
by  staining  methods  cannot  be  said  to  be  satisfactory.     By  staining 
in  a  saturated  aqueous  solution  of  fuchsin  in  the  cold  for  five  to 
seven  minutes,   and  subsequently  decolorising  with  acid  alcohol 
(nitric  acid  1  part,  alcohol  10  parts),  it  is  stated  that  the  leprosy 
bacillus  is  stained,  the  tubercle  bacillus  not. 


The  Smegma  Bacillus1 

The  smegna  bacillus  is  an  organism  found  in  the  smegma 
praeputii,  between  the  scrotum  and  thigh,  and  between  the 
labia.  It  also  occurs  in  the  cerumen,  occasionally  on  the 
skin,  and  possibly  in  the  sputum. 

It  is  a  small  bacillus  resembling  the  tubercle  bacillus 
in  size  and  appearance,  and,  like  the  latter,  is  difficult 
to  stain,  but  when  stained  with  carbol- fuchsin,  retains 
the  colour  after  treatment  with  a  25  per  cent,  mineral 
acid  (Plate  X.  6)  ;  it  is  also  Gram-positive.  It  has, 
therefore,  to  be  distinguished  from  the  tubercle  bacillus 
in  certain  localities,  viz.  in  urine  and  about  the  external 
genitals.  It  is  non-inoculable  on  animals,  and  does  not 
usually  grow  in  primary  cultures  on  ordinary  media, 
but  can  be  isolated  by  the  use  of  blood- serum  or  nutrose- 
agar,  on  which  it  forms  delicate,  ropy  colonies.  After 
isolation  it  grows  freely  on  agar  as  a  thin,  slightly  brownish, 
creamy  layer,  in  which  the  bacilli  may  be  very  short  but 

1  See  Neufeld,  Arch.  f.  Hygiene,  xxxix,  p.  184;  Zeitschr.  /.  Hyg., 
Xxxix,  1901  ;  and  Moeller,  Centr.f.  BakL,  xxxi,  1902  (Originale),  p.  278. 


PLATE  X. 


a.  Leprosy.     Section  of  skin,      x  1500. 


b.  The  smegma  bacillus.     Smear  preparation  of  smegma. 
X  1500. 


THE  SMEGMA  BACILLUS  339 

retain  their  acid- fast  properties ;  on  potato  it  forms 
minute  (0-5-1  mm.)  greyish  colonies.  It  has  been  sug- 
gested that  the  syphilis  bacillus  of  Lustgarten  is  identical 
with  the  smegma  bacillus ;  neither  is  decolorised  by 
Lustgarten's  permanganate  method,  but  while  the  smegna 
bacillus  after  staining  is  with  difficulty  decolorised  by 
acid,  and  is  easily  decolorised  by  alcohol,  the  reverse  is 
the  case  with  Lustgarten's  bacillus. 

Staining  and  Differentiation 

Film  preparations  of  smegna  may  be  stained  in  exactly  the  same 
manner  as  for  tubercle,  after  treating  the  preparations  with  ether 
to  get  rid  of  fatty  material. 

The  urine  should  be  drawn  off  with  a  catheter  when  it  is  to  be 
examined  for  the  tubercle  bacillus  ;  this  will  generaUy  exclude  the 
smegma  bacillus.  Young  and  Churchman  l  conclude  that  the 
smegma  bacillus  is  a  scant  invader  of  the  male  urethra,  and  that 
by  washing  the  glans  and  irrigation  of  the  urethra  it  may  be 
eliminated  from  the  urine. 

If  there  is  reason  to  suspect  the  presence  of  the  smegma  bacillus 
when  staining  for  tubercle,  Bunge  and  Tranteroth  2  recommend 
that  the  film  specimens  should  be  treated  as  follows  : 

(1)  Immerse  in  absolute  alcohol  for  three  hours. 

(2)  Immerse  in  5  per  cent,  chromic  acid  for  fifteen  minutes. 

(3)  Stain  in  warm  carbol-fuchsin. 

(4)  Decolorise  in  25  per  cent,  sulphuric  acid  for  two  to  three 
minutes. 

(5)  Counter-stain  in  a  concentrated  alcoholic  solution  of  methyl- 
ene-blue  for  five  minutes. 

The  smegma  bacillus  will  be  decolorised  by  this  method  (see  also 
p.  329). 

Coles  recommends  (Journal  of  State  Medicine,  vol.  xii, 
1904,  p.  225)  the  following  staining  method  : 

(1)  Spread  thin  and  even  films  on  slides,  and  fix  by  heat,  in  the 
ordinary  way. 

1  Johns  Hopkins  Hospital  Rep.,  vol.  xiii,  1906,  p.  15. 

2  Fortschrit.  der  Med.,  xiv,   1896,  Nos.  23  and  24.      See  also  ibid. 
No.  9. 


340  A  MANUAL  OF  BACTERIOLOGY 

(2)  While  still  warm  from  the  heat  fixation  flood  with  filtered 
carbol-fuchsin  for  half  a  minute.     Again  warm  for  a  few  second 
over  the  flame  without  actual  boiling.     Allow  it  to  stand  and  stain 
for  seven  minutes. 

(3)  Wash  thoroughly  in  running  water,  and  then  decolorise  in 
either  of  the  following  solutions  : 

(a)  In  Pappenheim's  solution.1 — Place  the  preparation  in  a  wide- 
mouthed  bottle  containing  the  solution  for  not  less  than  four,  and 
not  longer  than  twelve,  hours.  Wash,  dry,  and  mount.  Tubercle 
bacilli  are  the  only  organisms  stained  red. 

(6)  In  Pappenheim's  solution  without  methylene-blue. — Proceed  as 
in  (a)  ;  wash  in  water  and  counter-stain  for  a  minute  in  weak 
aqueous  methylene-blue  solution.  The  tubercle  bacilli  are  biilliantly 
red. 

(c)  In  25  per  cent,  sulphuric  acid. — Pour  on  a  few  drops  of  the 
acid  and  allow  it  to  act  for  half  a  minute.  Pour  off,  and  then  place 
the  preparation  in  a  wide-mouthed  bottle  containing  the  acid  for 
not  less  than  sixteen  hours  and  not  more  than  twenty-four  hours. 
Wash  thoroughly,  counter-stain  with  weak  aqueous  methylene-blue. 
Tubercle  bacilli  are  the  only  bacilli  which  retain  the  red. 

Acid-fast  bacilli  in  milk  and  butter. — Numerous  acid-fast  bacilli 
have  been  obtained  from  milk  and  butter.  They  usually  grow 
freely  and  quickly  on  agar  and  on  gelatin  without  liquefaction, 
sometimes  as  a  creamy  layer,  sometimes  as  a  dry,  crinkled  film, 
which  may  be  pigmented  (yellow,  orange,  pale  brown  or  brick  red). 
Some  are  pathogenic  to  guinea-pigs  by  massive  intra-peritoneal 
inoculation  only,  producing  a  plastic  peritonitis,  but  not  nodules 
in  the  organs.  In  culture,  the  bacilli  are  acid-fast  and  occasionally 
resemble  B.  tuberculosis,  but  are  generally  thicker.  (See  Petri 
Arb.  a.  d.  Kais.  Gesundheitsamte,  xiv,  1897  ;  Rabinowitsch,  Zeitschr. 
f.  Hyg.,  xxvi,  1897  ;  Grassberger,  Munch,  med.  Woch.,  1899,  Nos.  11 
and  12  ;  Tobler,  ibid,  xxxvi ;  Swithinbank  and  Newman,  Bacteri- 
ology of  Milk  [Murray,  1903].) 

Grass  bacilli  and  mist  bacillus. — Moeller  isolated  from  a  grass 
(Phleum  arvense)  an  acid-fast  bacillus  which  he  termed  the  Timothy- 
grass  bacillus  ;  other  grasses  also  yield  acid-fast  bacilli  (Grass 
Bacillus  II).  They  grow  readily  on  culture  media,  and  are  not  so 
acid-fast  as  the  tubercle  bacillus.  The  Mist  bacillus  was  isolated 
from  dung,  and  is  considered  by  Pettersson  to  be  identical  with 
the  Timothy-grass  bacillus.  (See  Moeller,  Deutsch.  med.  Woch., 

1  Pappenheim's  solution  consists  of  one  part  of  corallin  (rosolic  acid) 
in  100  parts  of  absolute  alcohol,  to  which  methylene-blue  is  added  to 
saturation  ;  20  parts  of  glycerin  are  then  added. 


GLANDERS  341 

1898,  p.  376  ;    Herr,  Zeitschr.  f.  Hyg.,  xxxviii,  1901  ;    Pettersson, 
Berl  klin.  Woch.,  1899,  p.  562.) 


Glanders  l 

Glanders  is  a  disease  which  has  been  known  from  the 
earliest  times,  being  recognised  by  the  Greek  and  Roman 
writers,  by  whom  it  was  termed  yuaX*?  and  malleus  respec- 
tively. It  is  distinctly  a  disease  of  the  horse,  mule,  and 


FIG.  39. — Nasal  septum  of  glandered  horse,  showing  ulceration  of 
Schneiderian  membrane  (McFadyean). 

ass,  but  is  also  communicable  to  man  and  to  certain 
other  animals.  It  is  caused  by  a  small  bacillus  discovered 
by  Loffler  and  Schiitz  in  1882. 

In  the  horse  the  lungs  are  always  affected,  and  fre- 
quently the  nasal  mucous  membrane  (Fig.  39).  Nodules 
form  which  afterwards  break  down  and  ulcerate,  and  a 
muco-purulent  discharge  appears  ;  in  the  older  writings 
the  name  "  glanders  "  covered  only  these  advanced  cases 
of  the  disease.  In  "  farcy  "  the  lymphatic  vessels  and 

1  See  McFadyean,  Journ.  of  State  Med.,  vol.  xiii,  1905,  pp.  1,  65,  and 
125. 


342 


A  MANUAL  OF  BACTERIOLOGY 


glands  are  affected,  the  enlarged  glands  being  known  as 
"  farcy  buds  "  (Fig.  40). 

In  man  the  disease  is  rare,  an  average  of  four  deaths 
per  annum  being  caused  by  it  in  this  country.  It  occurs 
in  two  forms — the  acute  and  the  chronic.  The  former 
is  a  very  serious  affection,  accompanied  by  high  fever, 
prostration,  and  delirium,  and  almost  invariably  fatal  in 
from  two  to  three  weeks.  The  seat  of  infection  is  usually 


FIG.  40. — Horse  affected  with  farcy  (McFadyean). 

the  hand  or  arm,  the  nasal  mucous  membrane  being 
sometimes  subsequently  involved,  and  deposits  may  form 
in  the  lymphatic  glands,  internal  organs,  and  muscles. 
In  the  chronic  form  intramuscular  abscesses  are  frequent, 
from  the  breaking  down  of  which  indolent  ulcers  may 
result ;  the  disease  runs  a  prolonged  course  of  weeks  or 
even  months,  and  about  half  the  cases  end  in  recovery. 
In  the  early  stage  an  eruption  may  develop  on  the 
forehead  and  face  simulating  very  closely  that  of 
smallpox, 


BACILLUS  MALLEI  343 

The  Glanders  Bacillus 

The  glanders  bacillus  (B.  mallei)  is  an  obligatory  parasite 
with  the  equine  species  for  its  normal  host.  It  hardly 
grows  on  artificial  media  below  about  20°  C.,  and  probably 
cannot  maintain  a  saprophytic  existence  outside  the 
animal  body. 

Morphology. — The  glanders  bacillus  occurs  in  the 
tissues  as  a  cylindrical  rod  with  rounded  ends,  varying 
between  2  //,  and  5  /m  in  length,  and  generally  straight, 
though  sometimes  slightly  curved.  The  bacilli  are  usually 
irregularly  scattered,  and  do  not  tend  to  form  colonies. 
In  stained  preparations  they  often  appear  more  or  less 
beaded,  or  may  exhibit  bipolar  staining,  but  some  stain 
uniformly.  The  bacilli  from  young  cultures  not  more 
than  twenty- four  hours  old  are  almost  always  short  rods, 
a  little  thicker  than  those  found  in  the  lesions  (Plate  XI.  a). 
In  old  broth  cultures  the  surface  growth  is  largely  com- 
posed of  filaments,  which  do  not  show  any  regular  seg- 
mentation, but  may  exhibit  lateral  branching,  and  may 
have  club-shaped  extremities.  From  these  features  some 
have  inferred  that  the  glanders  organism  belongs  to  the 
Streptothricce.  The  bacillus  does  not  form  spores,  and  is 
probably  non- motile,  though  in  a  hanging- drop  prepara- 
tion a  very  active  Brownian  movement  is  present. 

Staining  reactions. — The  bacillus  is  Gram- negative,  and 
is  not  acid-fast,  but  from  young  cultures  stains  readily 
with  the  ordinary  anilin  dyes.  In  smears  of  glanders  or 
farcy  material,  a  simple  staining  with  any  of  the  basic 
anilin  dyes,  with  subsequent  decolorisation  with  dilute 
acetic  acid,  suffices  to  demonstrate  it  if  it  is  present  in  any 
number,  a  difficulty  in  recognising  the  organism  being  the 
presence  of  deeply  staining  nuclear  detritus.  In  sections, 
methylene-blue  staining  with  decolorisation  in  dilute 
acetic  and  mordanting  with  tannin  gives  the  best  results 


344  A  MANUAL  OF  BACTERIOLOGY 

(p.  350).  The  bacillus  shows  dark  staining  dots  when 
treated  with  osmic  acid,  suggesting  fat- globules  (Shattock). 

Cultural  characters. — The  Bacillus  mallei  is  an  aerobic, 
and  facultatively  anaerobic  organism.  The  growth  on 
gelatin  at  22°  C.  is  scanty  and  pale  brownish  in  colour 
without  liquefaction.  On  glycerin  agar  it  forms  a  thick 
cream-  or  slightly  brown- coloured  growth,  and  on  blood- 
serum  a  somewhat  amber- coloured  growth,  which  after- 
wards becomes  brownish.  The  growth  on  potato  at 
37°  C.  is  most  characteristic,  and  practically  diagnostic. 
If  the  surface  of  the  potato  is  inoculated  with  a  loopful 
of  farcy  pus  or  material  from  the  centre  of  a  glanders 
nodule,  the  resulting  growth  is  usually  not  distinctly 
visible  until  the  third  day,  when  raised,  translucent, 
viscid,  amber- yellow  coloured  growth  or  colonies  appear. 
With  continued  incubation  the  colonies  coalesce,  the 
growth  becomes  thicker  and  fawn-coloured,  then  reddish- 
brown,  and  finally  generally  chocolate-brown.  The 
growth  is  also  odourless,  limited  to  the  site  of  implanta- 
tion, and  does  not  stain  the  potato.  Broth  or  glycerin 
broth  becomes  uniformly  turbid,  and  after  a  week  or  so 
patches  of  a  whitish  surface  scum  form,  and  after  three 
weeks  the  broth  is  nearly  covered  with  this  surface  growth, 
which  is  slimy  and  easily  broken  up  on  shaking.  Broth 
cultures  give  the  indole  reaction.  Litmus  glucose  agar 
becomes  pink.  Milk  is  not  coagulated. 

Resistance  to  Germicides.,  etc. — The  glanders  bacillus  is 
but  little  resistant,*  and  cultures  frequently  die  out  in  a 
month  or  so.  Complete  desiccation  at  37°  C.  of  nasal 
discharge,  farcy  pus,  or  bacilli  from  cultures,  is  frequently 
fatal  in  twenty- four  to  forty- eight  hours.  Young  broth 
cultures  are  soon  destroyed  by  bright  sunlight,  and  an 
exposure  of  ten  minutes  to  a  temperature  of  55°  C.  is  fatal 
to  the  cultivated  bacilli.  A  3  per  cent,  solution  of  carbolic 
acid,  a  1  per  cent,  solution  of  potassium  permanganate, 


PLATE  XI. 


a.  The  glanders  bacillus.     Film  preparation  of  a 
pure  culture,      x  1000. 


Section  of  a  glanders  nodule,  showing  giant -cells  (after 
McFadyean). 


PATHOGENICITY  OF  GLANDERS  BACILLUS    345 

and  a  1  in  5000  solution  of  corrosive  sublimate  are  fatal 
in  two  to  five  minutes. 

Pathogenicity ,  etc. — The  glanders  bacillus  varies  con- 
siderably in  virulence,  and  under  continued  cultivation 
may  become  almost  non-pathogenic. 

Glanders  is  met  with  exclusively  among  horses,  asses, 
and  mules,  and  man  is  infected  from  these  animals,  nearly 
all  cases  of  human  glanders  being  among  ostlers,  grooms, 
and  coachmen,  and  the  usual  mode  of  infection  is  by 
farcy  pus  or  nasal  discharge  coming  into  contact  with  a 
cutaneous  wound  or  abrasion.  A  remarkable  immunity, 
however,  is  enjoyed  by  the  slaughterers,  who  have  to  deal 
with  the  carcases  of  glandered  animals,  and  who  might 
be  supposed  to  run  the  greatest  risk.  But  it  must  be 
remembered  that  Babes  frequently  found  at  the  post- 
mortem on  persons  who  had  to  do  with  horses,  and  who 
died  from  diseases  other  than  glanders,  encapsuled  glanders 
nodules  in  the  lungs  and  internal  organs,  suggesting  that 
the  disease  may  often  be  latent  in  man,  who  appears  to 
be  relatively  insusceptible,  and  that  infection  may  be 
possible  by  inhalation.  In  the  horse  glanders  is  readily 
transmissible  experimentally  both  by  ingestion  and  by 
inoculation,  and  ingestion  is  probably  the  common  mode 
of  infection  naturally,  infection  by  inhalation  occasionally 
occurring.  Even  when  glanders  bacilli  are  administered 
experimentally  by  the  mouth  in  the  horse,  the  lesions  may 
be  most  prominent  in,  or  even  be  confined  to,  the  lungs. 
In  the  horse,  the  disease  has  periods  of  epidemic  prevalence, 
and  is  particularly  frequent  in  London.  In  1892  there 
were  3000  equine  cases  in  Great  Britain,  in  1903  there  were 
2499  cases,  and  nearly  90  per  cent,  of  all  cases  occur  in  the 
Metropolitan  area.  These,  it  is  to  be  noted,  were  cases 
in  which  the  disease  was  well  developed  and  manifest,  but 
there  are  also  numerous  others  in  which  it  is  latent. 
Guinea-pigs  and  field  mice  are  highly  susceptible  to  the 


346  A  MANUAL  OF  BACTERIOLOGY 

disease,  which  may  also  be  contracted  by  some  of  the 
Carnivora,  such  as  the  cat,  lion,  and  tiger,  by  inoculation 
or  by  feeding  on  diseased  carcases.  The  rabbit,  sheep, 
and  dog  are  but  slightly  susceptible,  while  cattle,  swine, 
and  house  mice  are  stated  to  be  immune.  Shattock1  found 
that  the  white  mouse  is  somewhat  susceptible,  and  suggests 
that  in  all  probability  the  house  mouse  is  similarly  so. 

In  the  horse  the  most  constant  seat  of  glanders  lesions 
is  the  lung,  and  McFadyeaii  states  that  no  case  of  glanders 
with  lesions  elsewhere  than  in  the  lungs,  and  with  these 
organs  unaffected,  has  ever  been  recorded.  In  nearly 
every  case  of  farcy,  also,  nodules  are  present  in  the  lungs. 
The  lung  lesions  have  the  form  of  rounded,  firm,  or  shotty 
nodules.  The  number  present  is  variable,  rarely  less  than 
a  dozen ;  exceptionally  there  are  hundreds,  fairly  evenly 
distributed  throughout  the  lung  tissue.  The  nodule 
commences  as  a  collection  of  polymorphonuclear  leucocytes, 
around  which  a  zone  of  congestion  is  present.  Later,  the 
alveolar  walls  undergo  necrosis,  and  the  leucocytes  necrose 
and  disintegrate,  but  their  chromatin  persists  as  rounded 
fragments  which  retain  their  affinity  for  nuclear  stains 
(chromatotaxis).  The  nodule  may  become  surrounded 
with  a  layer  of  thin  fibrous  tissue,  between  which  and 
the  necrotic  central  area  a  zone  of  endothelioid  cells  with 
giant- cells  may  be  present  (Plate  XI.  6). 

The  lesions  of  farcy  are  at  the  onset  histologically 
identical  with  the  glanders  nodule,  but  by  the  progressive 
liquefaction  of  the  tissues  actual  abscesses  form. 

The  lesions  set  up  in  an  inoculated  guinea-pig  are  very 
characteristic,  and  can  be  used  for  diagnostic  purposes. 
With  a  very  virulent  culture,  such  as  can  be  obtained  by 
several  passages  through  a  susceptible  animal,  a  guinea-pig 
may  die  in  four  or  five  days,  and  the  post-mortem  lesions 
are  slight,  consisting  of  some  caseation  at  the  seat  of 
1  Trans.  Path.  Soc.  Lond.,  vol.  lix,  1898,  p.  333. 


STRAUS'S  TEST  347 

inoculation  and  slightly  enlarged  spleen,  which  contains 
a  few  small  yellowish  nodules  resembling  miliary  tubercles. 
The  material  from  human  cases  as  a  rule  seems  more 
virulent  than  that  from  the  horse,  and  death  of  the  guinea- 
pig  often  ensues  a  few  days  after  inoculation. 

The  culture  or  material  from  a  glandered  horse  does  not 
usually  produce  death  of  a  guinea-pig  until  a  lapse  of 
two  or  three  weeks.  A  male  guinea-pig  being  chosen,  the 
changes  observed  are  caseation  followed  by  ulceration 
at  the  seat  of  inoculation,  when  this  is  done  subcutaneously, 
and  great  enlargement  of  the  testicles  ;  on  cutting  into 
these  they  are  found  to  be  partially  or  almost  entirely 
converted  into  a  pasty  caseous  material,  while  the  skin 
covering  them  is  so  adherent  that  it  can  only  be  detached 
by  cutting,  and  the  spleen  is  very  much  enlarged  and 
studded  with  small  yellowish  nodules.  In  a  female 
guinea-pig  the  ovaries  are  attacked.  These  appearances 
are  of  importance  in  the  diagnosis  of  the  disease.  The 
difficulty  of  finding  the  bacillus  in  the  discharges  by 
microscopical  and  staining  methods  is  so  great  that  these 
cannot  be  employed  with  any  certainty.  Loffler  and 
Straus  therefore  recommend  the  inoculation  of  a  male 
guinea-pig  intraperitoneally  with  the  discharge  or  other 
material.  If  the  glanders  bacillus  is  present  the  lesions 
thus  described  rapidly  ensue,  and  the  diagnosis  is  estab- 
lished in  four  or  five  days  (Straus's  test1).  At  the  present 
time  the  inoculation  method  has  been  almost  entirely 
superseded  by  the  introduction  of  mallein,  the  former 
being  reserved  for  clinical  diagnosis  in  man. 

McFadyean  found  that  the  blood  of  a  glandered  animal 
produces  agglutination  or  clumping  of  the  glanders  bacillus 
similar  to  that  obtained  in  the  agglutination  (Widal)  test 
for  typhoid,  and  has  suggested  this  reaction  as  a  means 
of  diagnosis.  As  an  aid  to  the  clinical  diagnosis  of  the 

1  See  also  Nicolle,  Ann.  de  Vlnst.  Pasteur,  xx,  1906. 


348  A  MANUAL  OF  BACTERIOLOGY 

disease  in  man  it  is  doubtful  if  the  method  of  serum  diagnosis 
can  be  applied,  for  Foulerton  found  that  typhoid  and  diph- 
theria sera  also  produce  agglutination  of  the  glanders  bacillus. 

Toxins.' — Mallein,  a  preparation  analogous  to  tuberculin, 
is  prepared  by  growing  a  virulent  glanders  bacillus  for  a 
month  or  six  weeks  in  glycerin  veal- broth  in  flat  flasks 
such  as  are  employed  for  tuberculin  (Fig.  38),  so  that  there 
is  free  access  of  oxygen.  The  culture  is  then  autoclaved 
for  fifteen  minutes  at  115°  C.,  filtered  through  a  Berkefeld 
filter,  concentrated  to  one  fourth  of  its  volume,  and  mixed 
with  an  equal  volume  of  a  J  per  cent,  solution  of  carbolic 
acid.  This  yields  an  active  mallein,  1  c.c.  of  which  is  a 
dose,  and  gives  a  good  reaction.  Like  tuberculin,  it 
possesses  feeble  curative  properties,  though  a  few  cases 
of  cure  by  prolonged  use  have  been  reported  by  Babes 
and  others,  but  is  used  for  diagnostic  purposes ;  the 
veterinary  authorities  are  unanimously  agreed  that  it  is 
one  of  the  most  certain  means  we  possess  for  diagnosing 
glanders  in  the  horse.  Injected  into  an  unglandered 
horse  little  or  no  effect  is  produced,  but  in  a  glandered 
animal,  about  twelve  hours  after  injection,  the  tempera- 
ture rises  1-5°  to  3°  C.  above  the  normal,  a  large  and 
painful  swelling  forms  at  the  seat  of  inoculation  (it  may  be 
as  large  or  even  larger  than  half  a  cocoanut),  while  any 
affected  lymphatic  vessels  or  farcy  buds  become  swollen. 
Reaction  may,  however,  be  produced  in  the  absence  of 
glanders  if  the  horse  is  being  treated  with  bacterial  products, 
toxins,  etc.1 

Epizootic  lymphangitis  has  a  superficial  resemblance  to 
farcy  in  the  horse,  and  must  not  be  mistaken  for  the  latter 
(see  "  Sporotrichosis,"  Chapter  XVI). 

The  greatest  care  should  be  exercised  when  working  with 
glanders    material    or    cultures,    several    fatal     laboratory 
accidents  having  unfortunately  happened. 
1  See  Sudmersen  and  Glenny,  Journ.  of  Hygiene,  vol.  viii,  1908,  p.  14. 


DIAGNOSIS  OF  GLANDERS  349 

Whit  more1  describes  a  glanders -like  disease  occurring  in  man  in 
Rangoon.  A  non-Gram-staining  bacillus  is  present,  morphologically 
like  the  glanders  bacillus,  but  killing  guinea-pigs  with  septicaemic 
symptoms  and  not  affecting  the  testes,  growing  well  and  luxuriantly 
on  culture  media,  liquefying  gelatin  slowly,  growing  well  on  potato 
with  at  first  a  cream-coloured,  and  subsequently  a  yellowish  growth, 
curdling  milk  and  not  fermenting  any  sugar. 


Clinical  Examination 

(1)  Prepare  and  stain  film  preparations  of  the  pus  or  discharge 
in  Loffler's  blue,  with  subsequent  partial  decolorisation  in  4  per 
cent,  acetic.     The  ordinary  pyogenic  cocci  will  not  be  found  unless 
a  secondary  infection  has  occurred,  and  the  material  may  appear 
sterile,  for  the  glanders  bacilli  may  be  very  scanty. 

(2)  Several  tubes  of  glycerin-agar  and  potato  should  be  inoculated 
and  incubated  at   37°   C.   for  seventy-two   hours.     On  the   agar, 
colonies  of  the  glanders  bacillus  will  develop  in  twenty-four  to 
thirty-six  hours,  but  the  potato  will  not  show  the  characteristic 
amber-yellow  growth  under  forty-eight  to  seventy-two  hours. 

(3)  It  will  usually  be  necessary  (in  man,  at  least)  to  confirm  the 
diagnosis  by  an  inoculation  experiment.     A  fully  developed  male 
guinea-pig  is  chosen,  and  a  little  of  the  discharge,  or  an  emulsion 
of  the  material  (O5  to  1  c.c.)  is   injected  intraperitoneally,  if   the 
material  be  fairly  sterile,  but  if  not,  subcutaneously.     In  three  to 
five  days  the  animal  should  show  the  characteristic  swelling  of  the 
testicles  if  the  material  be  glandered. 

(4)  An  ophthalmo-reaction  is  stated  to  be  reliable  both  in  man 
and  in  animals. 

(5)  In  animals  the  mallein  test  may  be  applied.     The  dose  is 
injected  subcutaneously  in  the  neck  over  the  vertebrae,  and  midway 
between  the  jaw  and  the  shoulder. 

(a)  The  temperature  of  the  animal  should  be  taken  if  possible 
morning  and  evening  for  two  or  three  days  previous  to  inoculation  ; 
in  any  case  at  the  twentieth  hour  after  inoculation,  or,  better,  at 
frequent  intervals  from  the  twelfth  to  the  twentieth  hour. 

(6)  A  complete  reaction  comprises  (i)  a  rise  of  temperature  of 
more  than  2-5°  F.,  (ii)  an  extensive  hot  and  painful  swelling  at  the 
seat  of  inoculation.     Systemic  disturbance,  such  as  prostration,  loss 
of  appetite,  shivering,  etc.,  majr  occur. 

(c)  The  temperature  reaction  is  unreliable  in  all  cases  in  which 

1  Journ.  of  Hyg.,  xiii,  1913,  p.  1. 


350  A  MANUAL  OF  BACTERIOLOGY 

the  temperature  at  the  time  of  inoculation  is  2-5°  F.  above  the 
normal.  In  such  cases,  if  there  be  any  suspicious  clinical  signs  to 
assist,  reliance  may  be  placed  upon  the  local  swelling. 

(6)  In  animals  the  agglutination  reaction  is  stated  by  Moore 
and  Taylor  l  to  give  accurate  results.     In  man  this  test  might 
give  an  inconclusive  result  (see  ante). 

(7)  In  the  tissues  the  glanders  bacillus  is  difficult  to  demonstrate. 
Sections  may  be  stained  for  half  an  hour  with  carbol  methylene- 
blue,  treated  with  4  per  cent,  acetic  for  a  few  seconds,  washed,  and 
rapidly  dehydrated  with  alcohol,  cleared  and  mounted.     McFadyean 
recommends,  after  treating  with  acetic  and  washing,  flooding  with 
a  saturated  solution  of  tannic  acid  in  water  for  fifteen  minutes, 
washing,  counter-staining  in  a  1  per  cent,  aqueous  solution  of  acid 
fuchsin  for  fifteen  to  thirty  seconds,  washing,  dehydrating,  and 
clearing  in  cedar  oil. 

Twort's  method  may  also  be  employed  (see  section  on  Amoeba 
coli,  "  Clinical  Diagnosis  "). 

1  Journ.  of  Infect.  Diseases,  Sup.  No.  3,  May  1907,  p.  85. 


CHAPTER  X 

TYPHOID  FEVER— PARA-TYPHOID  FEVER— BACILLUS 
ENTERITIDIS  AND  THE  GARTNER  GROUP— SWINE 
FEVER— BACILLUS  DYSENTERIC— BACILLUS  COLI 

THE  organisms  considered  in  this  chapter  form  a  natural  group  or 
family,  the  "  Typhoid-Colon  "  group,  and  pass  as  it  were  by  grada- 
tions in  cultural  characters  from  the  typhoid  bacillus  to  the  colon 
bacillus.  Loffler  classes  them  together  in  a  family,  the  Typhacese, 
which  is  divided  into  sub-families  :  (a)  Typheae,  which  includes  the 
B.  typhosus  and  B.  dysenteries ;  (b)  losarceae,1  which  includes  the 
Gartner  group  of  organisms  ;  and  (c)  Colese,  the  B.  coli  group  of 
organisms. 

The  group  can  be  divided  into  lactose  fermenters  and  non-lactose 
fermenters.  The  former  includes  B.  coli  and  its  variants.  There 
is  also  a  group  of  late  lactose  fermenters  (after  six  days)  which  occur 
in  the  intestine,  e.g.  B.  coli  mutabilis.  The  non-lactose  fermenters 
are  classified  by  Henderson -Smith  2  as  follows  : 

I.  Certain  groups  of  no  known  pathogenic  importance.     Frequent 
in  the  intestine. 

II.  The  Typhoid  group,  B.  typhosus. 

III.  Paratyphoid-Enteritidis  (Gartner)  group. 

1.  Atypical  members. 

a.  Saccharose  fermenters.     Not  agglutinated  with  Gartner 

or  paratyphoid  serum. 
6.  Dulcitol  non-fermenters. 

c.  B.  paratyphosus  A. 

d.  Salicin  fermenters.     Frequent  in  animals* 

2.  Typical  members. 

a.  B.  enter  itidis  of  Gartner. 

b.  B.  paratyphosus  B. 

c.  B.  suipestifer. 

1  From  los,  poison,  and  <rap£,  flesh. 

2  Centr.f.  Bakt.  Abt.  I  (Orig.),  68,  1913,  p.  151  (Bibliog.). 

351 


352  A  MANUAL  OF  BACTERIOLOGY 

IV.  Dysentery  group. 

1.  Mannitol  non-fermenters.     B.  dysenterice,  Shiga. 

2.  Mannitol  fermenters. 

a.  B.  dysenterice,  Strong. 

b.  Sorbite  fermenters. 

(a)  Dextrin  non-fermenters. 

(b)  Dextrin  fermenters. 

c.  Sorbite  non-fermenters. 

(a)  Dextrin  non-fermenters. 

(b)  Dextrin  fermenters. 

a.  Maltose  fermenters.     B.  dysenterice,  Flexner. 
/3.  Maltose  non-fermenters.     B.  dysenterice  Y. 

The  typhoid  bacillus  is  a  remarkably  stable  and  well-defined 
organism  showing  little  or  no  variation,  unlike  most  other  members 
of  the  group. 

All  the  foregoing  are  non-liquefiers  ;  for  convenience  certain 
liquefying  forms,  e.g.  B.  cloacce,  may  be  placed  in  this  group. 


Typhoid  Fever 

The  specific  organism  of  typhoid  fever  is  a  bacillus  origi- 
nally isolated  by  Eberth  in  1880,  and  more  closely  studied 
by  Gafiky  in  1884. 

The  Eberth- Gaff ky  bacillus,  or  Bacillus  typhosus,  is  best 
observed  in  sections  of  the  spleen,  in  which  it  occurs  in 
groups  or  colonies  consisting  of  short  rods  with  rounded 
ends,  each  measuring  about  3  jm  in  length.  It  has  also 
been  demonstrated  in  the  mesenteric  glands  and  liver,  in 
the  swollen  Peyer's  patches  before  ulceration,  and  in 
other  situations. 

Pure  cultivations  may  be  obtained  from  the  spleen 
during  life  by  puncture  (p.  370),  from  the  blood  (p.  369), 
sometimes  from  the  urine,  or  from  the  spleen  of  a  cadaver. 
In  the  latter  case  the  organ  is  washed,  and  then  cauterised 
lineally  by  means  of  a  red-hot  iron,  in  order  to  destroy 
the  saprophytic  bacteria  on  and  near  the  surface.  An 
incision  is  made  with  a  sterilised  knife  through  this 


BACILLUS  TYPHOSUS  353 

cauterised  area,  and  a  little  of  the  splenic  pulp  is  taken  with 
a  sterilised  platinum  needle  and  inoculated  on  to  tubes 
or  plates,  preferably  of  litmus  lactose,  Conradi-Drigalski, 
or  malachite-green,  agar.  These  are  incubated  at  37°  C. 
for  twenty-four  to  forty-eight  hours,  and  the  growths 
which  develop  are  examined  microscopically  and  are  tested 
by  agglutination  and  by  cultural  methods.  The  Bacillus 
typhosus  has  the  following  characters  : 

Morphology. — Bacilli  with  rounded  ends  averaging  3  u. 
in  length,  and  0-6  //.  broad.  It  is,  however,  in  cultivation 
a  markedly  pleomorphic  organism,  and  very  short  rods, 
long  rods,  and  thick  filaments  10  to  30  /u.  in  length  occur ; 
the  latter  are  known  as  involution  forms  (Plate  XII.  a). 
It  does  not  form  spores,  but  granulation  and  vacuolation 
may  be  observed  in  the  protoplasm,  particularly  in  old 
cultures. 

It  is  actively  motile,  and  possesses  a  number  of  flagella, 
arranged  peritrichically  both  at  the  poles  and  sides  (Plate 
XII.  c).  The  flagella  are  long  and  wavy,  and  average 
eight  to  twelve  in  number,  a  point  of  differentiation  from 
the  Bacillus  coli,  which  usually  has  only  three  or  four.  It 
stains  by  the  ordinary  anilin  dyes,  but  not  by  Gram's 
method. 

Cultural  characters. — The  B.  typhosus  is  aerobic  and 
facultatively  anaerobic,  and  grows  well  on  the  ordinary 
culture  media.  On  agar  it  forms  a  thick,  moist,  greyish 
layer.  On  gelatin  it  grows  slowly,  and  the  growth,  which 
is  usually  scanty  and  confined  to  the  needle-track,  is  white 
and  shining,  and  somewhat  irregular  (Plate  XII.  6).  The 
colonies  in  gelatin  are  visible  in  about  forty-eight  hours, 
and  form  small  roundish- white  points,  which  are  granular 
and  brownish  in  colour  by  transmitted  light.  In  broth 
it  produces  a  general  turbidity,  without  film  formation. 
The  growth  on  potato  acid  in  reaction  is  somewhat  charac- 
teristic ;  it  forms  a  moist,  grey,  shining  layer,  which  is 

23 


354  A  MANUAL  OF  BACTERIOLOGY 

almost  invisible.  If,  however,  the  reaction  of  the  potato  is 
neutral  or  alkaline,  the  growth  may  be  yellowish.  The 
B.  typhosus  grows  well  in  milk,  with  slight  permanent 
acidity,  but  without  coagulation. 

Acid  is  formed  in  small  quantity  during  its  growth  in 
many  media  (volatile  fatty  acids,  and  lactic  acid),  which 
can  be  demonstrated  by  cultivating  in  litmus  milk,  or  in 
litmus  glucose  media,  and  the  organism  will  grow  in  slightly 
acid  media.  Neither  gas  nor  indole  1  is  formed  in  cultures  ; 
acid  is  produced  from  glucose,  but  no  gas ;  lactose  is 
unacted  upon.  The  fermentation  reactions  on  various 
media  are  given  in  the  Table  on  p.  381,  and  are  there  con- 
trasted with  those  of  the  B.  coli  and  other  organisms  (see 
also  p.  384).  Chatterjee  2  finds  that  agar  on  which  the 
typhoid  bacillus  has  been  grown  contains  substances  which 
inhibit  further  development  of  the  organism  if  it  be  inocu- 
lated on  to  an  agar  culture  which  has  been  scraped  so  as 
to  remove  all  growth. 

Pathogenicity. — In  cases  of  typhoid  fever  in  man  the 
Bacillus  typhosus  is  widely  distributed  in  the  body,  in  the 
various  tissues,  and  in  the  blood,  from  which  it  may  be 
obtained  by  cultivations  made  from  at  least  0-5  c.c.  (see 
"  Clinical  Diagnosis,"  p.  369).  The  bacillus  is  constantly 
present  in  the  blood  from  the  commencement  of  the  disease, 
though  not  in  large  numbers,  and  cultures  from  the  blood 
in  competent  hands  result  in  the  recovery  of  the  organism 
in  approximately  100  per  cent,  of  the  cases  ;  in  the  later 
stages  of  the  disease  it  is  less  frequently  recovered.3  In 
addition  to  being  present  in  the  Peyer's  patches,  mesenteric 
glands,  and  spleen,  the  B.  typhosus  has  been  found  in  the 
rose-spots  of  the  eruption,  in  the  sweat,  in  the  sputum 

1  Occasionally  a  feeble  indole  reaction  may  be  obtained   by  careful 
testing. 

2  Trans.  Fourteenth  Internal.  Cong,  of  Hygiene  (Berlin,  1907),  Bd.  iv, 
p.  34. 

3  Coleman  and  Buxton,  Amer.  Journ.  Med.  Sci.,  June  1907. 


PLATE  XIL 


a.  Bacillus  typhosus.    Film  preparation  of  a 
pure  culture.      X  1500. 


6.  Gelatin  culture  of  B. 
typhosus,  six  days  old. 


c.  Bacillus  typhosus.     Film  preparation  showing 
flagella.     x  1500. 


BACILLUS  TYPHOSUS  355 

and  lungs  in  the  pulmonary  complications,  and  in  the 
urine.  In  the  urine  it  is  so  frequently  present  that  special 
disinfection  should  be  practised,  more  particularly  during 
convalescence,  and  in  some  cases  it  may  be  so  abundant  as 
to  produce  a  turbidity  (typhoid  bacilluria)  and  cystitis. 
It  is  also  pyogenic,  and  occurs  (usually  in  pure  culture)  in 
concurrent  or  post-typhoidal  complications,  e.g.  empyema, 
abscesses,  osteomyelitis,  suppurating  ovarian  cysts,1  etc. 
Clumps  of  bacilli  in  the  gall-bladder  have  been  suggested 
as  the  nuclei  of  gall-stones,  and  the  bacilli  may  be  so 
numerous  in  the  gall-bladder  and  bile-ducts  as  to  cause 
cholecystitis  and  cholangitis.  It  is  not  easy  to  isolate  the 
organism  from  the  stools,  and  plate  cultivations  on  special 
media  must  be  employed,  e.g.  Conradi-Drigalski,  malachite- 
green,  or  brilliant- green,  agar  (see  "  Water  "). 

Injected  intraperitoneally  into  mice  and  guinea-pigs 
the  B.  typhosus  usually  produces  death,  and  the  same 
result  follows  from  intravenous  injections  in  rabbits,  but 
the  pathogenic  effects  so  obtained  are  not  specific.  By 
continuous  cultivation  it  loses  its  pathogenic  properties. 
Given  by  the  mouth  no  result  follows,  and  the  same  is 
the  experience  of  most  observers  who  have  fed  animals  on 
typhoid  stools  ;  a  disease  process  analogous  to  typhoid 
fever  in  man  has  rarely  been  induced  experimentally. 
Remlinger 2  states  that  by  feeding  young  rabbits  on 
vegetables,  cabbage,  etc.,  soaked  in  water,  to  which  had 
been  added  some  culture  of  the  typhoid  bacillus,  he  has 
succeeded  in  inducing  a  condition  resembling  typhoid  fever 
in  man.  The  charts  which  accompany  the  paper  show 
a  typical  rise  of  temperature,  a  period  of  pyrexia  with 
morning  remission,  followed  by  a  typical  fall  of  tempera- 
ture. The  animals  suffered  from  diarrhoea,  and  their 
blood  gave  the  agglutination  reaction.  Post  mortem,  the 

1  Taylor,  Jcurn.  Obstet.  and  Gyncecol.  Brit.  Empire,  Nov.  1907. 

2  Ann.  de  rinst.  Pasteur,  xi,  1897,  p.  822. 


356  A  MANUAL  OF  BACTERIOLOGY 

intestine  was  congested  and  filled  with  yellow  diarrhoeic 
matter,  the  Peyer's  patches  were  swollen  and  in  some 
places  commencing  to  ulcerate.  The  spleen  was  increased 
to  two  or  three  times  its  normal  size,  and  cultures  of  the 
typhoid  bacillus  were  obtained  from  it.  MetchnikofE 1 
has  infected  the  chimpanzee  per  os  with  typhoid  faeces. 

The  proof  of  the  causal  relation  of  the  Bacillus  typhosus 
to  enteric  fever  is  based  on  the  following  facts.  It  is  met 
with  in  the  tissues  in  cases  of  enteric  fever,  can  be  obtained 
from  the  spleen  during  life  by  puncturing  with  a  hollow 
needle,  and  may  be  isolated  from  the  urine  and  blood 
during  the  course  of  the  disease,  and  is  not  met  with  in 
other  diseases.  The  writer  has  had  under  his  care  three 
cases,  and  knows  of  several  others,  in  which  the  disease 
was  almost  certainly  contracted  in  the  laboratory  from 
working  with  pure  cultures.  The  blood  and  blood-serum 
of  an  animal  immunised  against  the  B.  lyphosus  are  found 
to  bring  about  cessation  of  movement  and  agglutination 
or  aggregation  of  the  bacilli  in  a  broth  culture  of  the 
organism.  A  similar  result  occurs  when  the  serum  of  a 
patient,  in  the  second  week  of  an  attack  of  typhoid  fe^er, 
acts  on  the  B.  typhosus,  the  reaction  not  occurring  with 
healthy  individuals  or  in  other  diseases  (Plate  XIII.  a). 
This  indicates  that  in  the  body  of  an  individual  suffering 
from  typhoid  fever  the  same  substances  are  formed  as 
in  an  animal  artificially  immunised  by  cultures  of  the 
B.  typhosus.  This  reaction  is  now  recognised  as  a  valuable 
clinical  test  in  doubtful  cases  of  enteric  fever  (the  "  Widal  " 
or  agglutination  reaction  2). 

The  agglutination  reaction. — For  the  method  of  carrying 
out  the  agglutination  reaction  see  p.  190.  Normal  serum 
will  generally  agglutinate  the  typhoid  bacillus  in  a  dilution 

1  See  Ann.  de  VInst.  Pasteur,  xxv,  1911,  p.  193. 

2  Some  controversy  has  arisen  as  to  the  discoverer  of  this  reaction. 
Griinbaum  claims  to  have  first  observed  it. 


THE  AGGLUTINATION  REACTION          357 

up  to  1  in  3  or  4,  but  occasionally  is  more  active.  Dead 
bacilli  may  be  used.  The  reaction  is  not  obtained  before 
the  sixth  or  seventh  day  of  fever,  occasionally  not  until 
much  later.  Very  rarely  the  reaction  seems  to  be  inter- 
mittent. The  blood  may  retain  its  agglutinating  power 
for  years  after  an  attack,  and  inoculation  with  anti- 
typhoid vaccine  also  confers  agglutinative  properties. 
Cases  do  occur  in  which  agglutination  is  absent  throughout, 
but  they  are  rare  and  often  tend  to  be  severe  and  to  ter- 
minate fatally.  Usually,  if  the  blood  during  the  course 
of  an  attack  fails  to  give  a  reaction  when  tested  on  three 
occasions  at  intervals  of  three  to  four  days,  it  is  improbable 
that  the  case  is  one  of  typhoid  fever.  Moreover,  cases 
occur,  simulating  typhoid  closely,  due  to  infection  with 
the  so-called  para-colon  or  para-typhoid  bacilli.  These 
"  para  "  bacilli  belong  properly  to  the  Gartner  group  of 
organisms  (see  p.  371).  If  a  positive  reaction  be  obtained,  yet 
the  case  does  not  seem  to  be  one  of  typhoid,  a  previous  attack 
or  inoculation  with  typhoid  vaccine  must  be  excluded.  The 
previous  injection  of  a  typhoid  anti-serum  into  the  patient 
might  induce  a  non-typhoid  infection  to  give  the  reaction. 

Gwyn  1  found  that  out  of  265  cases  diagnosed  as  typhoid 
and  accurately  studied,  only  one  persistently  failed  to 
give  the  reaction.  The  blood  of  this  case,  however,  reacted 
typically  with  a  Gartner-like  organism  obtained  from  the 
blood  (a  case,  therefore,  of  para- typhoid  infection). 

Johnson  and  McTaggart  2  found  that  typhoid  blood  dried 
for  sixty  days  still  gave  a  typical  agglutination  reaction. 
An  incomplete  reaction  was  occasionally  obtained  as  early 
as  the  end  of  the  second  day,  and  the  complete  reaction 
was  rarely  delayed  beyond  the  fifth  day.  They  also 
noticed  that  the  blood  of  the  horse  often  produced  clump- 
ing, etc.,  of  typhoid  bacilli,  indistinguishable  from  an 

1  Johns  Hopkins  Hosp.  Bull.,  vol.  viii,  1900,  p.  387. 

2  Brit.  Med.  Journ.,  1896,  vol.  ii,  p.  629. 


358  A  MANUAL  OF  BACTERIOLOGY 

agglutination  reaction  with  typhoid  blood  ;  but  the  same 
agglutinating  effect  was  also  produced  on  the  colon  bacillus. 
Many  chemical  substances  also  produce  agglutination  of 
typhoid  bacilli,  so  that  it  is  necessary  to  exclude  them  in 
making  a  diagnosis.  For  example,  corrosive  sublimate 
(0-7  :  1000),  alcohol,  salicylic  acid,  vesuvin,  and  safranin 
(1  :  1000)  agglutinate,  while  carbolic  and  lactic  acids, 
chloroform,  caustic  soda,  and  ammonia  do  not,  the  two  last 
only  provided  the  test  typhoid  emulsion  be  made  with  dis- 
tilled water.  Safranin  has  a  powerful  agglutinating  action 
on  the  typhoid  bacillus,  but  not  on  the  colon  bacillus. 

While  there  is  no  constant  connection  between  the 
activity  of  agglutination  and  the  severity  of  the  disease, 
active  agglutination  tends  to  go  with  cases  which  recover, 
and  cases  in  which  agglutination  is  feeble  or  absent  tend 
to  be  severe. 

Toxins. — From  cultures  of  the  typhoid  bacillus  Brieger 
isolated  a  base  which  he  termed  typhotoxin,  and  which  is 
isomeric  with  gadinine.  In  animals  it  produced  salivation, 
profuse  diarrhoea,  paralysis,  and  death.  Brieger  and 
Frankel  isolated  from  cultures  a  toxic  protein  body. 
Fen  wick  and  Bokenham l  extracted  from  spleens  of 
typhoid  fever  patients  a  proteose,  an  alkaloid,  and  a  fatty 
residue.  The  proteose  produced  fever,  anorexia,  and  loss 
of  weight  in  guinea-pigs  and  rabbits,  but  the  alkaloid  and 
fatty  matter  were  without  effect. 

The  toxins  of  the  typhoid  bacillus,  however,  seem  to 
be  largely  intra- cellular,  and  filtered  broth  cultures  are 
usually  almost  non-toxic.  Sidney  Martin  2  by  cultivating 
in  a  protein  medium  was  able  sometimes  to  obtain  a 
toxic  nitrate,  a  few  c.c.  of  which  produced  lowered  tem- 
perature, diarrhoea  and  death.  Macfadyen  and  Rowland,3 

1  Brit.  Med.  Journ.,  1895,  vol.  i,  p.  801. 

2  Ibid.  1898,  vol.  ii,  pp.  11  and  73. 

3  Centr.  f.  Bakt.,  xxx,  p.  753. 


TYPHOID  CARRIERS  359 

by  disintegrating  large  quantities  of  typhoid  bacilli,  filter- 
ing, and  so  obtaining  the  intracellular  constituents  in 
the  filtrate,  found  that  small  doses  of  the  latter  produced 
a  transient  rise  of  temperature  in  guinea-pigs  and  a  loss 
of  weight  which  was  soon  recovered  from.  Animals  so 
treated  were  protected  against  a  certain  lethal  dose  of 
typhoid  bacilli,  and  their  blood  exhibited  agglutinative 
and  bacteriolytic  properties  towards  the  typhoid  bacillus. 
Macfadyen  *  later  obtained  the  intra- cellular  juice  of 
typhoid  bacilli  by  disintegration  after  freezing  with  liquid 
air,  and  found  it  to  be  very  toxic  to  guinea-pigs  by  intra- 
peritoneal,  and  to  rabbits  by  intra-venous  inoculation. 
The  writer  found  that  cultures  of  the  Bacillus  typliosus 
do  not  give  the  "  diazo  "  reaction. 

Survival  of  the  typhoid  bacillus  in  the  body. — Bacilli  may 
persist  in  the  spleen  for  weeks,  in  the  gall-bladder  fol 
years,  and  in  suppurative  lesions  for  six  years  or  more. 
Foster  and  Kayser  obtained  pure  cultures  irom  the  gall- 
bladders of  seven  out  of  eight  cases,  and  in  2  per  cent,  of 
the  cases  this  "  cholecystitis  typhosa  "  becomes  a  chronic 
process,  and  typhoid  bacilli  may  be  discharged  into 
the  bowel  for  long  periods.  Dean  2  found  this  to  be 
the  case  in  a  patient  who  had  had  enteric  fever 
twenty- nine  years  previously.  Such  "typhoid  carriers" 
have  been  the  subject  of  much  investigation  recently.3 
A.  and  J.  Ledingham  record  three  instances  met  with  in 
an  asylum  in  which  mysterious  cases  of  typhoid  had 
occurred — 31  cases  during  fourteen  years.  Davies  and 
Walker  Hall  4  relate  similar  outbreaks,  the  carrier  in  this 
case  being  a  woman  who  had  suffered  from  enteric  fever 
in  1901,  milk  serving  as  the  vehicle  of  transmission,  and 

1  Proc.  Roy.  Soc.  Lond.,  B.  Ixxi,  1902,  p.  77. 

2  Brit.  Med.  Journ.,  1908,  vol.  i,  p.  562. 

3  See   Ledingham,    Rep.    Med.    Off.    Loc.    Gov.    Board    for    1909-10 
(Bibliog.)  ;   ibid,  for  1912-13,  p.  336. 

4  Proc.  Roy.  Soc.  Med.,  vol.  i,  1908,  Epidemiolog.  Sect.,  p.  175. 


360  A  MANUAL  OF  BACTERIOLOGY 

a  number  of  other  instances  have  been  recorded.     Three- 
fourths  of  the  cases  are  women  (and  three-fourths  of  the 
cases   of  gall-stones   occur  in   women),   and  usually   the 
serum  of  the  carriers  gives  a  marked  agglutination  reaction, 
and  their  stools  frequently  contain  such  large  numbers  of 
typhoid   bacilli   that   these   largely   replace   the   natural 
bacterial  flora  of  the  intestine  and  may  often  be  recovered 
from  the  stools  by  simple  plating.     Firth's  statistics  give 
an  idea  of  the  frequency  of  the  development  of  the  carrier 
state.     Of  1229  cases  of  enteric  fever  among  the  British 
troops  in  India  bacteriologically  examined,   13   cases  of 
chronic  carriers  and  13  cases  of  temporary  carriers  were 
detected.     Obviously  the  typhoid  carrier  is  a  source  of 
serious  risk  to  the  community,  and  mysterious  outbreaks 
of  enteric  fever,  ascribed  by  some  in  the  past  to  a  "  de 
novo  "  origin  of  the  specific  organism,  become  explicable. 
Typhoid  convalescents  should  be  bacteriologically  examined 
before   discharge   from  hospital  and  the  negative   cases 
may  with  reasonable  safety  be  allowed  to  resume  their 
civil  life  (Ledingham).     The  typhoid  bacillus  may  occur 
in  the  contents  of  ovarian  cysts,  usually  causing  suppura- 
tion,   and   may   survive   for   months — twelve   in   a   case 
recorded  by  Taylor  l — after  the  attack  of  typhoid. 

Survival  of  the  typhoid  bacillus  outside  the  body. — The 
Bacillus  typhosus  has  been  isolated  in  a  few  instances  from 
WATER  SUPPLIES  which  have  become  infected,  and  have 
given  rise  to  epidemics,  as  in  the  case  of  the  Lincoln 
epidemic  in  1905. 2  This  is  the  exception,  however,  and 
the  isolation  of  the  typhoid  bacillus  from  an  infected  water 
is  a  very  difficult  matter  on  account  of  the  fact  that  the 
bacillus  may  have  died  out  before  the  investigation  is 
commenced,  that  it  is  generally  in  a  small  minority  and 
admixed  with  numbers  of  coliform  organisms,  and  that 

1  Journ.  Obstet.  and  Gyncecol.  Brit.  Empire,  November  1907. 

2  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1905-06. 


SURVIVAL  OF  BACILLUS  TYPHOSUS        361 

until  recently  no  medium  was  available  which,  inhibited 
the  growth  of  the  coliform  organisms  without  at  the 
same  time  inhibiting  the  growth  of  the  B.  typhosus.  By 
the  use  of  malachite  or  brilliant  green  media,  the  last- 
named  difficulty  seems  to  have  been  overcome  (see  section 
on  "  Water  "). 

In  sterilised  waters,  including  distilled  water,  the  Bacillus 
typhosus  maintains  its  vitality  for  upwards  of  a  month, 
and  in  some  cases  for  much  longer.  The  survival  is  not 
necessarily  longer  in  an  organically  polluted  water  than 
in  a  pure  water.  Infecting  sterilised  Thames  water  (from 
the  Temple  Embankment)  and  sterilised  tap-water  of  the 
Chelsea  Water-works  with  typhoid  cultures,  the  writer 
found  that,  examining  small  quantities  (1  c.c.)  of  the  water, 
the  bacillus  appeared  to  die  out  in  the  former  in  two  to  three 
weeks,  in  the  latter  in  four  to  five  weeks. 

The  survival  of  the  typhoid  bacillus  in  natural  waters 
must  be  influenced  by  many  circumstances — temperature, 
chemical  composition,  struggle  for  existence  with  the 
natural  bacterial  flora,  etc.,  of  the  water.  Experiments 
by  Russell  and  Fuller,1  in  which  the  organism,  suspended 
in  collodion  sacs,  was  subjected  to  the  action  of  lake  water, 
indicated  that  the  maximum  was  eight  to  ten  days. 
Houston,2  using  raw  Thames,  Lee,  and  New  River  waters 
artificially  infected  with  varying  quantities  of  ordinary 
laboratory  typhoid  cultures,  and  examining  quantities  of 
100  c.c.  of  the  water,  found  that  in  none  of  eighteen 
experiments  was  a  negative  result  obtained  in  four  weeks, 
and  it  was  only  after  nine  weeks  that  the  typhoid  bacillus 
could  not  be  isolated  from  this  quantity  in  all  the  experi- 
ments. But  in  subsequent  experiments,3  in  which  typhoid 
bacilli,  obtained  directly  from  the  urine  of  a  carrier  case 

1  Jown.  Infect.  Diseases,  Sup.  No.  2,  February  1902,  p.  40. 

2  First  Rep.  on  Research  Work,  Metropolitan  Water  Board,  1908. 

3  Sixth  Research  Report,  Metropolitan  Water  Board,  1911. 


362  A  MANUAL  OF  BACTERIOLOGY 

by  centrifuging  and  without  culturing,  were  added  to 
the  water,  the  number  of  bacilli  was  reduced  by  99'99  per 
cent,  after  a  week,  and  after  ten  days  the  organism 
could  not  be  isolated  from  100  c.c.  of  the  infected  water, 
indicating  that  the  uncultured  bacillus  rapidly  dies  in  a 
natural  water,  and  that  even  a  week's  storage  of  water 
affords  enormous  protection  against  water-borne  typhoid. 
In  aerated  (C02)  waters  the  B.  typhosus  does  not  survive  a 
fortnight.  The  methods  of  isolation  from  water  are  given 
in  Chapter  XXI. 

The  Bacillus  typhosus  may  gain  access  to  shell- fish,1 
oysters,  mussels,  cockles,  etc.,  particularly  if  obtained 
from  sewage-polluted  laying.  Such  polluted  shell-fish 
may  give  rise  to  typhoid  epidemics — as  at  Winchester 
and  Southampton  in  the  case  of  oysters,  and  in  the  case 
of  cockles,  derived  from  the  Thames  Estuary  and  imper- 
fectly cooked,  to  typhoid  cases.  Buchan  found  that  out 
of  855  primary  cases  of  typhoid  fever  occurring  in  house- 
holds in  Birmingham,  124,  or  14-5  per  cent.,  had  a  history 
of  mussel  eating,  and  in  seventeen  instances  the  histories 
were  conclusive  of  mussel  infection.  Mussels,  under 
certain  conditions  (which  are  not  well  understood),  are 
liable  to  develop  mytilotoxin,  etc.  (p.  38),  which  gives  rise 
to  gastro- enteritis.  Shell-fish  from  sewage-polluted  layings 
contain  B.  coli  in  varying  numbers,  but  from  uncontami- 
nated  layings  are  free  from  this  organism,  which  may 
therefore  serve  as  an  index  of  pollution  (see  "  Examination 
of  Shell-Fish,"  Chapter  XXI).  Contaminated  shell-fish, 
removed  to  pure  water,  gradually  cleanse  themselves — 
probably  after  two  to  three  weeks'  sojourn.  Klein  obtained 
the  typhoid  bacillus  from  artificially  infected  oysters,  kept 

1  On  pathogenic  organisms  in  shell-fish  see  Reports  by  Bulstrode  to 
the  Local  Government  Board,  1894  and  1911  ;  Rep.  Med.  Off.  Loc.  Gov. 
Board  for  1899-1900,  p.  574  ;  Houston,  Fourth  Report  of  the  Sewage 
Commission.,  vol.  iii,  1904  ;  McWeeney,  Loc.  Gov.  Board,  Ireland,  1904 ; 
Buchan,  Journ.  cf  Hygiene,  vol.  x,  1910,  p.  569. 


SURVIVAL  OF  BACILLUS  TYPHOSUS        363 

in  tanks  of  sea- water,  after  nine,  sixteen,  and  even  eighteen 
days  from  the  commencement  of  the  experiment,  the 
oysters  showing  no  abnormal  condition. 

As  regards  the  vitality  of  the  Bacillus  typhosus  in  sewage 
we  have  little  certain  information  ;  probably  it  tends  to 
die  out  within  a  few  days.  In  sterilised  sewage  inoculated 
with  it  the  B.  typhosus  hardly  multiplies  at  all,  and  at 
the  end  of  ten  days  has  died  out.  Certain  organisms  in 
sewage  seemed  to  have  a  deleterious  action  on  the  B. 
typhosus,  hastening  its  extinction,  viz.  the  B.  fluorescens 
Uquefaciens  and  B.  fluorescens  stercoralis.  Russell  and 
Fuller,  subjecting  the  bacillus  to  the  direct  action  of 
sewage,  found  the  survival  to  range  from  three  to  five 
days. 

In  dry  garden  earth,  according  to  Dempster,1  the  Bacillus 
typhosus  does  not  live  longer  than  eighteen  days  (Firth 
and  Horrocks  recovered  it  up  to  twenty- five  days),  and 
in  peat  it  dies  within  twenty-four  hours.  In  moist  soil, 
however,  the  bacillus  still  survived  on  the  forty-second 
day.  In  an  artificially  dried  soil  it  was  not  found  alive 
after  the  seventh  day. 

Sidney  Martin  found  that  in  moist  sterilised  soil  kept  at 
temperatures  from  3°  to  37°  C.,  the  B.  typhosus  maintains 
its  vitality  for  upwards  of  fifteen  months,  but  that  in 
unsterilised  soil  it  rapidly  dies.2 

Mair  3  concludes  that  the  typhoid  bacillus  can  survive 
in  natural  soil  in  large  numbers  for  about  twenty  days,  and 
is  still  present  in  a  living  condition  after  seventy  to  eighty 
days,  but  that  there  is  no  evidence  that  it  is  capable  of 
multiplying  and  leading  a  saprophytic  existence  in  ordinary 
soil.  He  suggests  that  Martin's  result  (the  rapid  extinc- 
tion of  the  bacillus  in  unsterilised  soil)  may  be  explained 

1  Med.-Chir.  Trans.,  vol.  Ixxvii,  1894,  p.  263. 

2  Reps.  Med.  Off.  Loc.  Gov.  Board  for  1896-1901. 

3  Journ.  of  Hygiene,  vol.  viii,  1908,  p.  37. 


364  A  MANUAL  OF  BACTERIOLOGY 

by  the  use  of  broth  cultures  for  infection,  the  broth  added 
causing  a  multiplication  of  the  saprophytes.  Firth  and 
Horrocks  l  similarly  conclude  that  the  typhoid  bacillus 
displays  no  tendency  to  increase  in  numbers,  nor  to  grow 
upwards  or  downwards  in  soil,  though  it  may  be  washed 
by  water  through  a  thickness  of  18  inches.  Neither  virgin 
nor  sewage- polluted  soils  differed  much  in  these  respects. 

Vitality  of  B.  typhosus  in  dust,  fomites,  etc. — Firth  and 
Horrocks  found  the  B.  typhosus  to  be  alive  in  soil  dry  enough 
to  form  dust  for  as  long  as  twenty-five  days,  and  consider 
that  infective  material  can  be  readily  transmitted  from 
dried  soil  and  sand  by  means  of  winds  and  air- currents. 
Doubtless  much  depends  on  the  degree  of  dryness  of  the 
substratum.  From  khaki  drill  and  serge  inoculated  with 
cultures  the  bacillus  was  recoverable  for  from  ten  to  twelve 
weeks,  and  for  from  ten  to  seventeen  days  from  the  same 
materials  fouled  with  enteric  faeces. 

Semple  and  Grieg,2  with  cloth  and  blanket  infected  with 
typhoid  urine,  failed  to  obtain  the  bacillus  after  seventeen 
days.  This,  however,  was  in  India,  and  the  survival  of 
the  typhoid  bacillus  on  fomites  probably  greatly  depends 
on  the  degree  of  drying  of  the  material.  A  striking  instance 
of  the  conveyance  of  infection  by  fomites  was  that  of  the 
blankets  used  in  the  South  African  War  and  brought  to 
this  country,  which  gave  rise  to  many  cases  of  typhoid 
fever. 

Firth  and  Horrocks  demonstrated  that  house-flies  can 
convey  enteric  infective  material  from  specific  excreta 
or  other  polluted  material  to  objects  on  which  they  settle 
or  feed,  and  the  Commission  which  investigated  the  preva- 
lence of  enteric  fever  in  the  Spanish- American  War  ascribed 
to  flies  the  principal  part  in  the  dissemination  of  the  disease 
(see  also  p.  389). 

1  Brit.  Med.  Journ.,  1902,  vol.  ii,  p.  936. 

2  Sc.  Mem.  Gov.  of  India,  No.  32,  1908. 


SEWER  GAS  AND  DISEASE  365 

There  has  always  been  considerable  discussion  on  the 
exact  relation  of  "  sewer-gas  "  to  disease.  It  is  generally 
held  that  sewer-gas  is  at  least  a  predisposing  cause  to 
enteric  fever,  diphtheria  and  tonsillitis.  Some  have 
considered  that  the  specific  organisms  are  present  in  the 
emanations  from  sewers,  and  this  may  occasionally  be 
the  case.  Thus  Horrocks,1  in  some  experiments  performed 
at  Gibraltar,  by  pouring  sewage  artificially  infected  with 
typhoid  culture  down  drains,  showed  that  specific  bacteria 
present  in  sewage  may  be  ejected  into  the  air  of  ventilation 
pipes,  inspection  chambers,  drains  and  sewers  by  (a)  the 
bursting  of  bubbles  at  the  surface  of  the  sewage,  (6)  the 
separation  of  dried  particles  from  the  walls  of  pipes, 
chambers  and  sewers,  and  probably  by  (c)  the  ejection  of 
minute  droplets  from  flowing  sewage.  "  Sewer- gas  "  may 
also  lower  vitality  and  increase  susceptibility.  Thus  Alessi 
found  that  animals  exposed  to  drain  emanations  are  at 
first  more  susceptible  to  infection,  but  after  a  month  or 
so  acquire  tolerance  and  are  no  more  susceptible  than 
animals  kept  under  ordinary  conditions.  Exposure  to 
the  gaseous  emanations  from  putrefying  matter  is  stated 
by  Trillat  to  increase  the  virulence  of  pathogenic  bacteria. 
There  is  no  evidence  that  sewer-men  or  those  employed  at 
sewage-works  suffer  from  ill-health. 

Action  of  heat,  germicides,  etc. — The  B.  typhosus  in  broth 
culture  is  killed  by  a  temperature  of  53°-54°  C.  in  half  an 
hour,  and  of  56°-60°  C.  in  ten  minutes.  It  is  readily 
destroyed  by  antiseptics.  (See  Table,  Chap.  XXII.) 

Semple  and  Grieg  (loc.  cit.)  found  bright  sunlight  to  be 
germicidal  in  from  two  to  six  hours. 

Wines  and  spirits  have  some  germicidal  action  on  the 
typhoid  bacillus.  Champagne  destroys  the  bacillus  in 
ten  minutes,  white  wines  in  fifteen  to  twenty  minutes, 
red  wines  in  thirty  minutes  or  thereabouts.  If  diluted 

1  Journ.  Roy.  San.  Inst.,  May  1907, j>.  176. 


MO  A  MANUAL  OF  BACTERIOLOGY 

with  water  the  germicidal  action  takes  much  longer  to 
accomplish,  and  the  acidity,  not  the  alcohol  content,  seems 
to  be  the  active  factor. 1  Spirits,  such  as  whisky  or  brandy, 
if  diluted  with  not  more  than  one  to  two  times  the  volume 
of  water,  kill  in  ten  to  twenty  minutes. 

Anti-typhoid  serum. — Attempts  have  been  made  to 
prepare  an  anti-typhoid  serum  by  inoculating  horses  with 
increasing  doses  of  typhoid  bacilli,  first  killed  (by  heat, 
chloroform,  etc.)  and  then  living,  but  such  sera  have 
proved  quite  useless. 

Macfadyen  2  prepared  an  endotoxic  serum  by  treating 
horses  with  the  endotoxin  obtained  by  triturating  the 
bacilli  in  the  presence  of  liquid  air.  The  writer  continued 
the  work,  and  obtained  a  serum  which  gave  promising 
results.3 

Chantemesse,4  by  cultivating  a  virulent  strain  of  the 
typhoid  bacillus  in  a  special  broth  made  with  ox  spleen, 
heating  the  culture  to  55°  C.,  centrifuging  and  injecting 
horses  with  the  fluid,  obtains  a  serum  which  he  claims  has 
marked  curative  properties,  the  mortality  being  4-3  per 
cent.,  as  against  17  per  cent,  for  those  subjected  to  ordinary 
treatment.  The  patients  receive  very  small  doses  of  the 
serum — five  or  six  drops — and  the  dose  is  repeated  only 
two  or  three  times.  This  dosage  is  quite  different  from 
that  of  an  ordinary  antitoxic  or  antimicrobic  serum,  and 
Wright  suggested  that  toxins  (and  not  anti-bodies)  in  the 
serum  may  be  the  active  agents.  Chantemesse  has 
accepted  this  view,  and  the  treatment,  therefore,  seems 
to  be  a  vaccine  one. 

1  Sabrazes  and  Marcandier,  Ann.  de  Vlnst.  Pasteur,  1907. 

2  Proc.  Ray.  Soc.  Lond.,    B,  vol.  Ixxi,  1903,  pp.  76  and  351  ;    Brit. 
Mcd.  Journ.,  1906,  vol.  i,  p.  905. 

3  See  Hewlett,  Goodall  and  Bruce,    Proc.   Roy.  Soc.  Med.,  vol.  ii, 
1907-08  (Medical  Section),  p.  245  et  seq.  ;  and  Hewlett's  Serum  Therapy, 
p.  220. 

4  Trans.  Fourteenth  Internat.  Cong.  Hygiene  and  Demography,  1907. 


ANTI-TYPHOID  VACCINE  367 

The  disease  has  also  been  treated  with  a  vaccine  (con- 
sisting of  a  killed  culture)  with  promising  results  by  Semple, 
Smallman,  Leishman,  and  others.  The  initial  dose  is 
40-100  millions,  and.  the  amount  is  cautiously  increased 
up  to  300-400  millions. 

Anti-typhoid  vaccine. — Wright  first  prepared  an  anti- 
typhoid vaccine  by  the  following  method.1  A  typhoid 
culture  of  moderate  virulence  (the  virulence  being  kept 
up  by  intraperitoneal  passage  through  guinea-pigs)  is 
grown  in  peptone  beef  broth  in  flasks  at  37°  C.  for  from 
fourteen  to  twenty-one  days.  The  flasks  are  then  so 
heated  that  their  contents  attain,  and  remain  at  for  a 
few  minutes,  a  temperature  of  60°  C.  To  obtain  uniform 
toxicity,  the  contents  of  several  flasks  should  be  mixed, 
and  to  safeguard  the  vaccine  from  contamination  one 
twentieth  of  its  volume  of  10  per  cent,  lysol  is  added. 
Various  ingenious  devices  have  been  adopted  by  Wright 
and  Leishman  to  prevent  contamination  and  for  stan- 
dardisation. 

The  immunising  power  of  a  typhoid  vaccine  depends 
upon  the  number  of  bacilli  it  contains,  and  on  the  particular 
strain  of  bacillus  used.  The  vaccine  is  standardised  by 
counting  the  number  of  bacilli  it  contains  by  Wright's 
method  (p.  220).  Leishman  2  now  cultivates  for  about 
forty-two  hours,  and  the  bacteria  are  killed  by  heating  to 
53°  C.  for  one  hour,  the  higher  temperature  having  proved 
to  be  deleterious,  and  after  cooling  0*25  per  cent,  of  lysol 
is  added  ;  it  is  not  necessary  to  employ  a  virulent  bacillus. 
In  the  early  days  the  symptoms  produced  by  the  inocula- 
tion were  often  severe,  but  with  more  moderate  methods 
are  now  hardly  appreciable.  Two  doses  of  the  vaccine 
should  be  given,  with  an  interval  of  about  ten  days  between 
the  two,  the  doses  being  500  and  1000  millions  respectively. 

1  Wright  and  Semple,  Brit.  Med.  Journ.,  1897,  vol.  i,  p.  256. 

.2  gee  Journ.  Roy.  Inst.  Pub.  Health,  vol.  xviii,  1910,  pp.  385,  440,  513. 


368  A  MANUAL  OF  BACTERIOLOGY 

The  vaccine  deteriorates  on  keeping.  Emulsions  of  agar 
cultures  and  autolysed  cultures  have  also  been  used  for 
preparing  vaccines. 

Inoculation  is  now  being  extensively  practised,  and 
Leishman  (loc.  cit.)  gives  the  following  statistics  of  its 
value  :  total  number  under  observation,  18,483-19,314  ; 
average  period  under  observation,  twenty  months  ;  number 
inoculated,  10,378 ;  number  uninoculated,  8936 ;  case- 
incidence  of  enteric  per  1000,  inoculated  5'39  ±  0*48, 
uninoculated  30*4  ±  1-23  ;  case-mortality  per  100,  inocu- 
lated 8'9,  uninoculated  16'9.  In  the  French  navy  Chante- 
messe  states  that  during  nine  months  in  1912,  among 
67,843  unvaccinated  persons  542  cases  of  typhoid  fever 
occurred,  while  among  3107  vaccinated  ones  not  a  single 
case  of  typhoid  occurred. 

Variation  of  the  B.  typhosus. — Allusion  has  already  been 
made  to  T wort's  work  on  the  "  education  "  of  B.  typhosus 
to  ferment  lactose,  and  on  the  apparent  conversion  of 
B.  typhosus  into  B.  alkaligines  by  Horrocks  (p.  6).  Penfold 
also  records  variations  in  the  fermentive  powers  of  B. 
typhosus  (Journal  of  Hygiene,  vol.  xi,  1911,  p.  30). 


Relapses 

Various  hypotheses  have  been  advanced  to  account  for  the 
relapses  which  occur  in  typhoid  and  other  diseases  (e.g.  Malta  and 
relapsing  fevers).  Chantemesse  and  Widal x  showed  that  if  the 
B.  typhosus  is  injected  into  an  animal  together  with  toxins  of  the 
streptococcus,  B.  coli,  or  Proteus,  its  virulence  is  enhanced,  or  the 
animal's  resistance  may  be  lowered.  If,  then,  immunising  and 
bactericidal  properties  of  the  blood  and  tissues  are  but  slightly 
acquired  during  the  attack,  an  absorption  of  toxic  substances  from 
the  alimentary  tract  may  be  sufficient  to  give  the  typhoid  bacilli 
still  present  a  fresh  start,  and  so  produce  a  relapse.  This  Sanarelli  2 
was  able  to  do  experimentally.  Wright  and  Lamb  formulated 

1  Ann.  de  VInst.  Pasteur,  vi,  1892,  p.  755. 

2  Ibid,  vi,  1892,  p.  721  ;  and  ibid,  viii,  1894,  p.  193. 


RELAPSES  369 

another  hypothesis.1  The  organisms  in  typhoid,  Malta,  and  re- 
lapsing fevers,  are  deposited  in  the  spleen  and  internal  organs, 
multiply  and  form  colonies  there,  which  become  protected  from 
the  bactericidal  substances  by  the  formation  of  a  non-anti-bacterial 
envelope.  When  the  anti-bacterial  substances  in  the  blood  and 
lymph  have  increased  to  such  an  extent  as  to  penetrate  and  abolish 
the  non-anti-bacterial  envelopes  which  surround  these  colonies, 
the  production  of  toxins  will  be  so  diminished  that  the  temperature 
will  fall.  If,  however,  for  some  reason  or  other,  even  a  single 
colony  escapes  the  full  anti-bacterial  power  of  the  lymph,  owing,  it 
may  be,  to  being  shut  off  in  a  capillary  which  has  become  blocked, 
or  in  some  other  part  not  freely  infiltrated  by  the  blood-  or  lymph- 
streams,  the  bacteria  of  this  colony  will  go  on  multiplying  until  the 
blood  has  become  modified  in  such  a  manner  as  to  bring  about  a 
diminution  of  the  anti-bacterial  substances,  and  thus  render  a 
relapse  possible. 

A  third  theory  has  been  suggested  by  Durham.2  He  regards  a 
given  infection  as  due  to  the  "  result  of  the  action  of  a  sum  of  a 
number  of  infecting  agents,  each  of  which  is  similar  but  not  identical 
in  its  nature,"  the  apparently  simple  infection  being  "  in  reality  a 
complex  phenomenon  brought  about  by  a  number  of  varieties  and 
sub-varieties  of  the  given  microbe."  He  suggests,  therefore,  that 
in  a  typhoid  infection  a  particular  race  of  typhoid  bacilli  is  in  excess, 
and  when  the  anti-bodies  for  this  particular  race  have  been  formed 
in  sufficient  quantity,  the  disease  process  comes  to  an  end.  There 
may,  however,  be  present  at  the  same  time  other  races  which  have 
produced  little  of  their  specific  anti-bodies  ;  these  then  begin  to 
grow  and  multiply,  and  a  relapse  ensues. 

In  the  case  of  relapsing  fever  the  organism  may  be  a  protozoon, 
and  in  protozoal  diseases  relapses  coincide  with  developmental  cycles 
of  the  parasite,  e.g.  in  malaria. 


Clinical  Diagnosis 

(1)  Blood  cultures. — Three  to  5  c.c.  of  blood  are  withdrawn 
from  a  superficial  vein  with  a  syringe  with  aseptic  precautions, 
and  0-5  c.c.  of  the  blood  so  obtained  is  sown  into  each  of  several 
tubes  containing  15  to  20  c.c.  of  sterile  broth.  The  tubes  are 
incubated  at  37°  C.,  and  if  organisms  develop  these  are  isolated  and 

1  Lancet,  1899,  vol.  ii,  p.  1727  ;  Sc.  Mem.  Med.  Officers  of  Ind.  Army, 
pt.  xii. 

2  Journ.  Path,  and  Bact.,  vol.  vii,  1901,  No.  2,  p.  240. 

24 


370  A  MANUAL  OF  BACTERIOLOGY 

examined  culturally  for  the  typhoid  bacillus.  Coleman  and  Buxton 
recommend  the  following  culture  medium  :  Ox-bile,  90  c.c.,  glycerin 
10  c.c.,  and  peptone  2  grm.  Distribute  in  small  flasks,  20  c.c.  in 
each,  and  sterilise.  Each  flask  is  inoculated  with  2  to  3  c.c.  of 
blood,  incubated  for  eighteen  to  twenty-four  hours,  then  streaks 
from  each  are  made  on  to  litmus  lactose  agar  plates,  which  are 
incubated  for  a  few  hours.  If  the  growth  does  not  redden  the  medium 
and  a  typhoid-like  bacillus  is  present,  it  is  tested  for  agglutination 
with  typhoid-immune  serum. 

(2)  Agglutination  reaction. — This  is  carried  out  by  the  micro- 
scopic or  the  macroscopic  (sedimentation)  method  described  at 
p.    190.     Dilutions  of  1  :  30,  1  :  50,  and  1  :  100  should  be  made. 
The  microscopic  method  is  the  more  rapid.     Various  apparatus 
(agglutinometers)  can  be  obtained,  consisting  of  measuring  devices 
and  a  supply  of  dead  culture,  with  which  the  sedimentation  test 
can  be  carried  out  by  any  one,  but  are  unsatisfactory  in  the  tropics. 

(3)  Ophthalmo-diagnosis. — Chantemesse  (loc.  cit.)  has  devised  a 
method   analogous    to    the   ophthalmo-diagnosis   for   tuberculosis 
(p.  330).     The  material  is  prepared  from  agar  cultures  of  typhoid 
which  are  emulsified,  dried,  triturated,  and  extracted,  and  the 
extract  is  precipitated  with  absolute  alcohol  and  dried  (for  details 
see   Hewlett's   Serum   Therapy  (p.    382).     The   dry  substance  is 
powdered  in  an  agate  mortar,  and  for  use  8  to  10  mgrm.  are  dissolved 
in  1  c.c.  of  sterile  water.     Of  this  solution  a  drop  is  instilled  into  the 
conjunctival  sac  ;  in  a  case  of  typhoid,  after  a  lapse  of  two  to  three 
hours  the  conjunctiva  becomes  red  and  there  is  a  sensation  of  heat, 
after  six  to  ten  hours  there  is  a  marked  conjunctivitis,  which  may 
persist  for  one  to  three  days  and  then  passes  off.     In  healthy 
persons  and  in  other  diseases  no  conjunctivitis  ensues.     A  cutaneous 
reaction  has  also  been  devised. 

(4)  Puncture  of  the  spleen  with  a  sterilised  hypodermic  needle  and 
syringe. — A  little  of  the  blood  and  pulp  is  withdrawn  with  the 
syringe,  and  cultivations  are  made  as  in  (1).     This  method  seems 
hardly  justifiable,  and  now  that  the  blood-culture  method  and 
agglutination  reaction  have  been  introduced  should  be  discarded. 

(5)  Examination  of  pus. — Cultivations  may  be  made  as  in  (1) 
if  the  bacillus  is  present,  apparently  in  pure  culture.     If  not,  plate 
cultivations,  preferably  on  litmus  lactose  agar,  Conradi-Drigalski, 
malachite-  or  brilliant-green  agar,  may  be  prepared  (see  "  Water  "). 

(6)  Examination  of  the  stools. — This  is  hardly  practicable  for 
clinical  diagnosis  ;   it  takes  too   long,  is    tedious  and  uncertain. 
Plate  cultivations  from  the  dilated  stools  are  made  on  Conradi- 
Drigalski,  malachite-  or  brilliant-green,  agar  (see  "Water"). 


THE  GARTNER  GROUP  371 


The  Gartner  or  Enteritidis  Group  of  Bacilli 

The  Gartner  group  of  bacilli,  of  which  the  type  is  the  B.  enteritidis 
of  Gartner,  are  bacilli  morphologically  resembling  the  B.  typhosus, 
i.e.  they  are  pleomorphic,  actively  motile,  •  multi-flagellate,  non- 
sporing,  and  non-Gram-staining,  but  culturally  are  intermediate 
between  B.  typhosus  and  B.  coli.  Thus,  like  B.  coli,  they  ferment 
glucose  with  the  production  of  gas  and  acid  and  change  neutral 
red  ;  like  B.  typhosus  they  do  not  attack  lactose  and  do  not  curdle 
milk.  In  litmus  milk  they  usually  first  produce  slight  acidity, 
followed  after  three  to  four  days  by  a  change  to  alkalinity,  and  the 
milk  ultimately  becomes  limpid.  The  fermentation  reactions  of 
some  members  of  the  Gartner  group  are  given  in  the  Table  on  p.  381. 
The  organisms  of  the  Gartner  group  may  be  divided  into  four  sub- 
groups : 

1.  Enteritidis  group. — Produce  acute  gastro-intestinal  disturbance 
in  man.     The  cause  of  epidemic  meat-poisoning,  e.g.  the  B.  enteritidis 
of  Gartner. 

2.  Pneumonic  group. — Produce  pneumonic  symptoms  in  man. 
The  cause  of  some  outbreaks  of  epidemic  pneumonia,  e.g.  B.  psitta- 
cosis. 

3.  Paratyphoid  group. — Produce   a  disease  resembling  typhoid 
fever  in  man.     May  also  produce  "food-poisoning"  with  gastro- 
enteritis.    Subdivisions  A  or  a  and  B  or  /3. 

4.  Group  non-pathogenic  to  man,  e.g.  B.  typhi  murium. 


The  Bacillus  enteritidis 

A  number  of  outbreaks  of  what  has  been  termed  "  epi- 
demic meat  poisoning "  have  been  traced  to  infection 
with  the  B.  enteritidis.  (See  also  "  Food  Poisoning," 
Chap.  XXL)  The  disease  takes  the  form  of  an  acute 
gastro- enteritis — urticaria,  abdominal  pain,  vomiting,  diar- 
rhoea, nervous  symptoms  and  collapse — occurring  from 
eight  to  thirty-six  hours  after  partaking  of  a  meat  meal, 
usually  pork  (sausage,  pork-pie,  ham),  occasionally  beef 
and  tinned  meat.  The  principal  outbreaks  of  this  nature 
have  been  those  at  Jena,  in  1888,  investigated  by  Gartner, 
and  from  which  he  isolated  the  type  form  of  the  B.  enteri- 


372  A  MANUAL  OF  BACTERIOLOGY 

tidis  ;  Welbeck  in  1880  ;  Middlesborough  in  1888  ;  Mans- 
field in  1896  ;  and  Derby  in  1902.  A  small  outbreak 
occurred  at  Bedford  in  1907.1  These  outbreaks  are  usually 
caused  by  varieties  of  the  B.  enteritidis  having  the  general 
characters  of  the  group,  which  usually  do  not  ferment 
lactose,  and  are  distinguishable  by  agglutination  reactions 
and  fixation  tests,  the  organism  isolated  as  a  rule  agglu- 
tinating well  with  the  patient's  serum. 

The  B.  enteritidis  in  morphology,  motility,  and  staining 
reactions  resembles  the  B.  typhosus,  forms  no,  or  only 
traces  of,  indole,  and  changes  neutral  red  to  a  fluorescent 
yellowish  colour.  Litmus  milk  after  a  faint  acidity  becomes 
alkaline,  and  is  converted  into  a  thin  watery  translucent 
fluid,  without  coagulation.  It  does  not  attack  either 
salicin  or  glycerin.  The  fermentation  reactions  are  given 
in  the  Table  on  p.  381.  Savage2  obtained  this  organism 
from  only  one  out  of  fifty-three  specimens  of  human 
excreta  examined.  A  number  of  variants  were  isolated 
from  various  materials,  some  fermenting  salicin,  some 
glycerin,  and  some  both  these  substances  (see  "  Meat," 
Chap.  XXI). 


Swine  Fever  or  Hog  Cholera3 

Swine  fever,  or  hog  cholera  (to  be  distinguished  from  swine 
erysipelas,  which  see),  is  an  infective  disease  of  pigs,  highly  con- 
tagious, and  causing  considerable  mortality.  The  duration  of  the 
affection  is  usually  three  to  four  weeks  ;  the  animals  lie  about, 
their  temperature  is  raised,  and  they  may  suffer  from  cough  and 
frequent  respiration,  and  some  lameness  in  the  hind  legs.  Towards 
the  end  mucous  diarrhoea  is  a  prominent  symptom.  Post  mortem, 
the  large  intestine  is  found  to  be  ulcerated,  the  ulcers  much 
resembling  the  typhoid  ulcers  of  man,  and  according  to  Klein, 

1  PuUic  Health,  vol.  xx,  1907-8,  p.  310. 

2  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1909-10,  p.  446. 

3  See    Uhlenhuth,    Trans.    Fourteenth    Internal.    Cong,    of   Hygiene 
(Berlin,  1907),  Bd.  iv,  p.  50  ;  Journ.  Roy.  Inst.  Pub.  Health,  1911. 


SWINE  FEVER  373 

pneumonia  is  commonly  present,  whence  he  termed  the  disease 
"  pneumo -enteritis."  McFadyean,  however,  from  his  own  experi- 
ence and  that  of  the  Board  of  Agriculture,  considers  pneumonia 
very  infrequent.  The  ulcers  occur  mainly  in  the  caecum  and  colon, 
and  are  due  to  a  well-defined  circular  necrosis  involving  the  whole 
thickness  of  the  mucous  membrane  and  occasionally  extending  to 
the  wall  of  the  bowel.  A  diffuse  diphtheroid  lesion  also  occurs,  due 
to  a  superficial  necrosis  with  deposition  of  a  thin  layer  of  fibrinous 
exudate  on  the  surface  of  the  mucous  membrane.  All  gradations 
are  found  between  the  well-defined  circular  necrosis  and  the  diffuse 
diphtheroid  lesion. 

An  organism  constantly  present  is  a  member  of  the  para- typhoid 
sub-group  of  the  Gartner  group  (B.  suipestifer  oisuicholerce,  apparently 
identical  with  B.  aertryck),  but  it  seems  to  be  a  terminal  infection 
and  not  the  true  ctiological  agent,  as  the  blood  and  tissues  filtered 
through  a  porcelain  filter  are  still  infective — i.e.  the  organism  is 
probably  ultra-microscopic.  Some  confusion  exists  in  the  nomen- 
clature of  the  disease.  Swine  fever  is  the  British,  and  hog  cholera 
the  American,  name.  In  addition,  a  disease  of  swine  was  formally 
described  under  the  designation  "swine  plague"  ("Schweine- 
seuche,"  Schiitz).  This  clinically  much  resembles  swine  fever,  but 
pneumonia  is  a  prominent  lesion,  and  a  non-motile,  stumpy, 
bi-polar  staining  bacillus  belonging  to  the  group  of  the  haemorrhagic 
septicaemia  bacilli  is  present  (see  under  "Chicken  Cholera"). 
This  is  now  regarded  as  a  secondary  infection  and  the  disease  as 
being  really  swine  fever.  The  B.  suipestifer  is  apparently  identical 
with  the  B.  ictero'ides  of  Sanarelli.  (See  also  Chap.  XIX.) 

Although  the  lesions  are  very  similar,  swine  fever  has  nothing  to 
do  with  typhoid  fever  of  man,  nor  with  ulcerative  colitis. 

Other  organisms  belonging  to  the  Gartner  group  are : 

1.  The  Danysz  bacillus,  used  as  a  virus  for  exterminating  rats 
(the  Danysz  virus). 

2.  The  B.  icteroides  of  Sanarelli,  supposed  by  him  to  be  the  cause 
of  yellow  fever,  but  apparently  identical  with  the  B.  suipestifer  (see 
"  Yellow  Fever,"  Chap.  XIX). 

3.  The  B.  typhi  murium  of  Loffler,  used  as  a  virus  for  exterminating 
mice. 

4.  The  B.  psittacosis  of  Nocard,  causing  an  infective  disease  of 
parrots  and  transmissible  to  man  (bird-fanciers,  etc.),  in  whom  it 
produces  a  severe  and  often  fatal  broncho-pneumonia. 

5.  Summer  diarrhoea. — Morgan  x  concluded  that  the  summer  or 

Ri  *  Brit.  Med.  Journ.,  1906,  vol.  i,  pp.  908  and  1131  ;  ibid.  1907, 
vol.  i,  p.  16. 


374  A  MANUAL  OF  BACTERIOLOGY 

epidemic  diarrhoea  of  infants  is  not  caused  by  the  dysentery  bacillus 
(see  p.  379).  In  50  per  cent,  of  the  cases  he  isolated  a  motile 
bacillus  producing  acid  and  gas  from  glucose  which  appears  to  be 
most  closely  allied  to  the  hog-cholera  bacillus,  differing  from  the 
latter  by  producing  alkalinity  in  litmus  milk  (without  previous 
acidity)  and  much  indole,  and  by  failing  to  produce  acid  and  gas 
from  mannitol,  arabinose,  maltose,  and  dextrin.  It  does  not  fer- 
ment dulcitol,  saccharose,  salicin  and  sorbite.  There  are  two 
variants,  designated  as  No.  1  and  No.  2.  Eyre  and  Minett 1 
examined  the  normal  faeces  of  sixty  young  children,  and  in  four 
only  isolated  a  bacillus  allied  to  the  Morgan  bacillus.  The  method 
of  isolation  was  by  means  of  plates  of  bile-salt  agar  containing 
1  per  cent,  of  mannitol  and  coloured  with  neutral  red.  (See  also 
Chap.  XX.) 

Para-typhoid  Fever  2 

The  name  "  para-colon  "  bacillus  was  given  by  Gilbert  in  1895 
to  races  of  bacilli  intermediate  in  type  between  the  typhoid  bacillus 
and  the  colon  bacillus,  and  this  designation  was  also  applied  by 
Widal  and  Nobecourt  to  a  bacillus  isolated  by  them  from  an 
abscess  in  the  neighbourhood  of  the  thyroid.  The  name  "  para- 
typhoid "  bacillus  appears  first  to  have  been  used  by  Archard  and 
Bensaude  in  1896,  and  was  reintroduced  by  Schottmiiller  in  1901, 
and  would  seem  to  be  the  preferable  designation  for  those  micro- 
organisms that  produce  typhoidal  symptoms. 

Para-typhoid  fever  may  be  defined  as  a  disease  much 
resembling  typhoid  fever  in  its  clinical  aspect,  which  is, 
however,  caused,  not  by  the  typhoid  bacillus,  but  by 
organisms  belonging  to  the  para-typhoid  sub-group  of  the 
Gartner  group  of  bacilli.  Para-typhoid  infections  some- 
times occur  in  epidemics,  may  be  spread  by  drinking- 
water  and  by  "  carriers,"  and  occur  in  all  parts  of  the 
world. 

Para-typhoid  bacilli  are  also  occasionally  the  pathogenic 
agents  in  cases  of  "  food  poisoning  "  with  gastro- enteritis, 
particularly  B.  suipestifer  (or  aertryck). 

1  Brit.  Med.  Journ.,  1909,  vol.  i,  p.  1227. 

2  See  Savage,  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1COS-9,  p.  316  ; 
JBainbridge  and  O'Brien,  Journ,  of  Hygiene,  vol.  xi,  1911,  p.  68  (Bibliog,). 


PARA-TYPHOID  FEVER  375 

The  para-typhoid  bacilli  are  morphologically  like  the 
typhoid  bacillus  and  are  actively  motile,  but  they  ferment 
glucose  with  the  production  both  of  acid  and  of  gas.  A 
number  of  races  have  been  isolated  differing  from  one 
another  in  their  source,  rate  of  fermentation  of  glucose, 
action  on  milk,  action  on  neutral  red,  and  agglutination 
reaction,  and  are  distinguished  by  the  names  of  those  who 
isolated  them. 

Two  groups  of  para- typhoid  bacilli  may  be  distinguished 
which  have  been  termed  A  and  B  by  Buxton.  Group  A 
produces  less  gas  in  glucose  media  than  group  B  ;  with 
group  A  milk  remains  permanently  acid  ;  with  group  B 
it  becomes  alkaline  after  a  transient  acidity  ;  and  though 
group  A  changes  neutral  red  to  yellow,  the  red  colour  tends 
to  return  after  three  weeks  or  so,  while  with  group  B  the 
yellow  colour  is  permanent.  That  is  to  say,  in  its  reactions 
group  A  is  more  closely  allied  to  the  typhoid  bacillus  than 
is  group  B. 

B.  para-typhosus  A  or  a  is  rarely  found,  the  vast  majority 
of  cases  of  para-typhoid  fever  being  associated  with  the 
presence  of  the  B  or  p  type.  The  fermentation  reactions 
of  some  of  the  para-typhoid  bacilli  are  given  in  the  Table 
on  p.  381. 

As  regards  the  agglutination  reaction,  the  blood  of  the 
para-typhoid  fever  patient  either  does  not  agglutinate 
the  typhoid  bacillus  or  agglutinates  it  only  in  low  dilution 
— e.g.  1  in  10  to  40,  while  it  agglutinates  the  para-typhoid 
bacilli  in  far  higher  dilution — e.g.  I  in  100  or  200,  or  even 
higher  ;  thus  in  Cushing's  case  the  patient's  serum  agglu- 
tinated the  para-typhoid  bacillus  isolated  from  it  up  to 
1  in  8000. 

The  diagnosis  of  para-typhoid  fever  would  be  based 
on  (a)  the  agglutination  reaction  ;  (6)  the  isolation  of  a 
para-typhoid  bacillus  by  cultures  from  the  blood  (p.  369). 
Prophylactic  vaccines  for  para-typhoid  fever  may  be 


376  A  MANUAL  OF  BACTERIOLOGY 

prepared  with  para-typhoid  bacilli  in  the  same  manner  as 
for  typhoid  fever  and  Castellani  has  made  use  of  a  mixed 
typhoid-para-typhoid  vaccine. 

Bacillus  dysenteriae  l 

In  one  type  of  dysentery,  the  so-called  epidemic  or 
bacillary  form  (see  "  Dysentery,"  Chap.  XX),  a  bacillus 
B.  dysenteries,  is  the  causative  agent.  The  B.  dysenteries 
includes  a  group  of  closely  allied  organisms. 

The  dysentery  bacillus  was  first  isolated  in  1897  by 
Shiga  in  Japan.  Somewhat  later  Kruse  isolated  an  almost 
identical  bacillus  in  Germany,  and  this  type  is  known  as 
the  Shiga-Kruse  type.  Later,  Flexner  and  Strong  isolated 
another  type  of  the  dysentery  bacillus,  and  during  the 
last  few  years  similar  organisms,  but  differing  from  the 
Shiga-Kruse  and  Flexner  types  in  some  of  their  fermenta- 
tion and  other  reactions,  have  been  isolated  ;  these  are 
sometimes  termed  "  pseudo-dysentery  "  bacilli. 

The  Shiga-Kruse  and  other  types  of  dysentery  bacilli 
have  been  isolated  by  Flexner  and  Strong  in  the  Philip- 
pines, Park,  Duval,  Bassett,  Martini,  Hiss,  Russell  and 
others  in  the  United  States,  Castellani  in  Ceylon,  Rogers 
and  others  in  India,  RufTer  and  Willmore  in  Egypt  (El 
Tor),  and  Eyre,  McWeeney  and  others  in  the  British  Isles. 

Morphology. — The  B.  dysenterice  are  small  slender  bacilli 
much  resembling  the  colon  bacillus.     They  are  non-motile, 
but  Brownian  movement  is  often  active,2  Gram-negative, 
and  non-sporing,  and  are  readily  destroyed  by  heat  (58° 
60°  C.)  and  antiseptics. 

Cultural  characters. — The  dysentery  bacilli  are  aerobic 
and  facultatively  anaerobic.  On  agar  a  thinnish  creamy 

1  See   an  excellent   summary   by   P.   H.    Bahr,   Dysentery  in   Fiji 
(Witherby  &  Co.,  London,  1912). 

2  Flagclla  have  been  described  by  some  observers,  but  cannot  usually 
be  demonstrated. 


BACILLUS  DYSENTERIC  377 

growth  develops  ;  on  gelatin  a  white  growth  nearly  limited 
to  the  inoculation  track,  and  without  liquefaction.  The 
colonies  on  a  gelatin  plate  closely  resemble  those  of  the 
typhoid  bacillus.  On  potato  the  growth  is  either  thin, 
grey  and  slightly  visible,  or  thicker  and  yellowish  or 
brownish.  The  colour  of  neutral  red  media  is  unaltered. 
Litmus  milk  first  becomes  faintly  acid,  then  markedly 
alkaline ;  no  clotting.  Indole  is  generally  not  formed 
(never  by  the  Shiga  type)  ;  occasionally  a  trace  may  be 
detected.  All  strains  ferment  glucose  with  the  formation 
of  acid  only,  no  gas  ;  none  ferment  lactose.  Some  strains 
(the  Flexner  type)  ferment  mannitol  with  the  formation 
of  acid  only,  no  gas  ;  other  strains  (the  Shiga-Kruse  type) 
have  no  action  on  this  alcohol.  The  principal  fermenta- 
tion and  other  reactions  are  given  in  the  Table  on  p.  381. 
These  reactions  are  very  variable  with  different  stains, 
but  differentiation  may  be  accomplished  by  agglutination, 
saturation,  and  complement  fixation,  tests.  Shiga x  dis- 
tinguishes five  groups  of  dysentery  bacilli  as  follows  : 

1.  Fermenting  dextrose  alone  [Shiga,   Kruse,  Flexner 

(Newhaven)]. 

2.  Fermenting  dextrose  and  mannitol  (Hiss  and  Russell's 

Y  bacillus,  Ferran,  Seal  Harbour  bacillus). 

3.  Fermenting     dextrose,     mannitol     and     saccharose 

[Flexner,  Strong  (Manila)]. 

4.  Fermenting  dextrose,  mannitol,  maltose  and  saccha- 

rose (Harris,  Gay,  Woolstein). 

5.  Fermenting  dextrose  and  maltose,  and  giving  a  feeble 

acid  reaction  with  mannitol  (Shiga). 

Bahr  found  occasional  variations  in  fermentive  power 
after  sub-culturing  and  after  a  sojourn  in  flies. 

Agglutination  reaction. — The  agglutination  reaction  is 
given  by  the  blood  of  patients  suffering  from  the  bacillary 

1  Zeitsch.  f.  Hyg.,  Ix,  1908,  pp.  75,  120. 


378  A  MANUAL  OF  BACTERIOLOGY 

form  of  dysentery,  but  not  by  the  amoebic  form  (unless  a 
double  infection  be  present,  which  occasionally  is  the  case). 
The  agglutination  reaction  is  obtained  in  dilutions  of  1 
in  10  to  1  in  100,  but  may  occur  only  with  the  particular 
strain  causing  the  infection.1  Thus  by  the  agglutination 
reaction  variations  between  different  strains  of  the  B. 
dy  sentence  may  be  detected. 

Pathogenic  action. — The  organism  seems  limited  to  the 
bowel  and  its  mucous  membrane  and  does  not  gain  access 
to  the  blood.  No  characteristic  lesions  are  produced  in 
animals  by  administration  of  the  dysentery  bacillus  per  os. 
In  man,  cultures  given  by  the  mouth  are  stated  to  have 
induced  a  typical  dysentery.  Animals  such  as  rabbits, 
guinea-pigs  and  mice  are  very  sensitive  to  injections  of 
living  and  killed  cultures  ;  in  fact,  it  is  very  difficult  to 
immunise  animals  against  the  organism.  Amounts  of 
0*1-0'2  mgrm.  of  an  agar  culture  given  intravenously 
or  intraperitoneally  are  fatal  to  these  animals. 

In  man  the  organism  is  abundant  in  the  bloody  mucoid 
discharge  from  the  bowel,  and  at  an  early  stage  is  easy  to 
isolate  by  means  of  Conradi-Drigalski  agar  plates,  on  which 
it  forms  small  transparent  blue  colonies  ;  at  a  later  stage 
(after  two  to  three  days)  the  other  organisms  in  the  bowel 
multiply  to  such  an  extent  that  isolation  may  become 
very  difficult.  "  Carriers  "  occur  and  help  to  spread  the 
disease,  which  may  be  conveyed  by  infected  water  and 
food  and  by  flies. 

Toxins. — The  nitrate  of  dysentery  cultures  (four  to  six 
weeks  old)  in  a  somewhat  highly  alkaline  broth  (broth 
just  alkaline  to  litmus  +  7  c.c.  normal  NaOH  per  litre)  is 
markedly  toxic,  O'l  c.c.  being  a  fatal  dose  for  a  large 
rabbit.2 

Anti-serum  and  vaccine. — The  serum  of  horses  immu- 

1  See  Hewlett,  Trans,  Path.  Soc.  Lond.,  vol.  Iv,  1904,  p.  51. 

2  Todd,  Journ.  of  Hygiene,  vol.  iv,  1904,  p.  480  (Bibliog.). 


THE  COLON  BACILLUS  379 

nised  with  the  toxin,  or  with  dead  and  then  with  living 
cultures,  possesses  marked  antitoxic  properties,  and  the 
use  of  this  antitoxic  serum  has  been  successful  in  cases 
of  acute  bacillary  dysentery.  Shiga  obtained  a  reduction 
in  mortality  from  22  to  7  per  cent,  by  the  use  of  serum 
in  a  severe  epidemic,  and  striking  results  were  obtained 
by  Buffer  and  Willmore1  in  Egypt  and  by  Bahr  in  Fiji. 
It  is  necessary,  however,  to  employ  a  serum  prepared  with 
the  particular  strain  of  the  disease. 

When  the  disease  has  become  chronic  the  use  of  a 
vaccine,  consisting  of  a  culture  sterilised  by  heat,  is  some- 
times beneficial.  Castellani  also  suggests  the  use  of  a 
vaccine  for  prophylactic  purposes. 

Para-dysentery  bacilli. — In  the  dysenteries  of  Ceylon, 
Castellani  2  has  sometimes  isolated  dysentery  bacilli  nearly 
related  to  the  Shiga-Kruse  type,  but  showing  differences 
from  it  in  agglutination,  persistence  of  acid  reaction  in 
litmus  milk,  and  virulence  ;  these  he  has  termed  "  para- 
dysentery  "  bacilli. 

Asylums  dysentery  and  summer  diarrhoea  of  infants.— 
Both  in  America  and  in  England  some  cases  of  summer 
diarrhoea  of  infants  are  found  to  be  associated  with  the 
B.  dysenteries  (see  above,  p.  374).  The  asylums  or  insti- 
tutional dysentery,  or  ulcerative  colitis,  is  also  due  to 
this  organism,  and  the  blood  of  patients  gives  the  agglu- 
tination reaction.3  In  both  instances  the  B.  dysenteries 
present  is  of  the  Shiga-Kruse  type. 


Bacillus  coli 

The  Bacillus  coli,  or  colon  bacillus  (B.  coli  communis), 
is  an  organism  of  considerable  importance,  both  in  con- 

1  Brit.  Med.  Journ.,  1909,  vol.  ii,  p.  862,  and  1910,  vol.  ii,  p.  1519. 

2  Journ.  of  Hygiene,  vol.  iv,  1904,  p.  495. 

3  Hewlett,  Trans.  Path,  Soc,  Lond,,  vol.  Iv,  1904,  p.  51. 


380  A  MANUAL  OF  BACTERIOLOGY 

nection  with  the  Bacillus  typhosus,  in  pathological  pro- 
cesses, and  in  water  supplies  as  an  indication  of  pollution. 
As  its  name  implies  it  is  a  constant  inhabitant  of  the 
intestinal  tract  in  man  and  animals  (except  perhaps  in 
certain  arctic  animals),  and  is  one  of  the  most  widely 
distributed  organisms  in  nature.  While  the  term  "  colon 
bacillus  "  is  applied  to  a  fairly  well-defined  organism  (the 
"  typical  B.  coli  "),  there  are  a  number  of  allied  organisms 
differing  from  the  type  in  one  or  more  characters — e.g. 
motility,  indole  production,  fermentation  reactions,  rate  and 
extent  of  milk  curdling,  etc. — and  these  varieties  are  said  to 
belong  to  the  "  colon  group,"  or  are  termed  "  coliform." 

The  B.  coli  may  be  readily  isolated  by  inoculating  litmus 
lactose  bile-salt  peptone-water  tubes  with  a  trace  of  a 
suspension  of  fresh  faeces,  growing  for  from  twenty-four 
to  forty- eight  hours  at  42°  C.,  and  plating  the  culture  on 
litmus  lactose  agar,  on  gelatin,  or  on  Conradi-Drigalski 
agar,  or  by  direct  plating  of  the  faeces  suspension  on  the 
last-named  medium  (see  also  "  Water  "). 

Morphology. — The  B.  coli  is  a  short  rod  with  rounded 
ends,  2  or  3  /x  long  and  0*5  /m  broad,  frequently  linked  in 
pairs  or  more.  It  is  often  so  short  that  it  is  merely  ovoid 
in  shape  ;  and,  on  the  other  hand,  longer  individuals  and  in- 
volution forms  occur  10  /UL  or  more  in  length  (Plate  XIII.  6). 
It  is  feebly  motile,  and  possesses  lateral  flagella  to  the 
number  of  three  or  four  on  an  average,  which  are 
shorter  and  straighter  than  those  of  the  typhoid  bacillus. 
It  is  sometimes  met  with  in  diplococcoid  form,  which  by 
cultivation  in  ascitic  fluid  may  become  fixed.  Capsulated 
forms  have  been  described. 

Spore-formation  does  not  occur,  but  vacuolation  may 
sometimes  be  observed.  The  organism  stains  well  by 
the  ordinary  anilin  dyes,  but  is  Gram-negative. 

Cultural  characters. — The  B.  coli  is  aerobic  and  faculta- 
tively anaerobic,  and  grows  readily  on  the  ordinary  culture 


FEKMENTATION  REACTIONS 


381 


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a. 


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I        I        I        I  I       I       I        I        I        I        I        I       + 


>IIIK  snui^ii 


*  ^* 


+    i     i 


I      1      I      I 


I  I       I       I        I        I       +      +       I       +      + 


I  I 


I      i     +    +    +    +    + 


SHI  I 

- 


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ftj          flq    cq      oq      Bq      cq      eq 


PQ      pq 


382  A  MANUAL  OF  BACTERIOLOGY 

media  from  20°  to  42°  C.  In  gelatin  plates  the  colonies 
are  visible  in  twenty-four  to  forty-eight  hours.  The  deep 
colonies  are  spherical,  granular,  and  of  a  pale  brownish 
colour,  darker  at  the  centre  than  at  the  periphery.  The 
superficial  colonies  are  at  first  punctate,  round  and  almost 
transparent,  but  subsequently  spread  on  the  surface  and 
may  attain  a  diameter  of  3  mm.,  their  margins  become 


FIG.  41. — Colonies  of  the  colon  bacillus,  superficial  and  deep. 

irregular,  the  surface  is  smooth,  they  are  finely  granular, 
opalescent  in  appearance,  and  are  thicker  at  the  centre 
than  at  the  periphery  (Fig.  41).  On  a  gelatin  streak  a 
copious  white,  shining,  smooth  growth  develops,  the  mar- 
gins of  which  are  irregular  and  crenated  (Plate  XIII.  c), 
and  in  old  cultures  the  medium  becomes  opalescent.  In 
a  gelatin  stab-culture  a  white  growth  develops  along  the 
line  of  inoculation  with  one  or  more  gas-bubbles.  The 
gelatin  is  not  liquefied.  On  agar  and  on  blood-serum  a 
thick,  moist,  shining,  greyish  layer  forms.  There  is 
abundant  formation  of  gas  in  a  stab-culture  in  glucose-agar 


PLATE  XIII. 


a.  The  agglutination  reaction.     A  clump  of  typhoid  bacilli. 
X  1500. 


b.  Bacillus  coli.    Film  preparation  from  a  pure        c  Gelatin  culture  of 
culture,      x  1000.  B.  coli,  six  days  old. 


THE  COLON  BACILLUS  383 

and  in  gelatin  shake  cultures  (Fig.  42),  provided  the  latter 
medium  be  made  with  meat ;  "  lemco  "  gelatin,  however, 
generally  fails  to  give  gas.  On  acid  potato  it  forms  a 
straw-yellow  or  brownish-yellow,  moist,  thick  growth,  but 
if  the  potato  is  not  fresh  and  acid  in  reaction  the  growth 
may  be  colourless.  Milk  is  a  good  culture  medium,  and 
is  curdled  in  twenty- four  to  seventy- two  hours.  This 


FIG.  42. — Colon  bacillus.     Gelatin  shake  culture  showing  gas 
production. 

curdling  is  principally  due,  not  to  an  enzyme,  but  to  the 
formation  of  a  considerable  amount  of  lactic  acid,  though 
a  milk-curdling  enzyme  has  been  described  by  Savage  1 
as  being  formed  under  certain  conditions.  The  gas  which 
is  produced  in  culture  media  under  anaerobic  conditions 
consists  of  hydrogen  and  carbon  dioxide.  Under  aerobic 
conditions  marsh  gas  is  stated  to  be  also  formed.  The 
ratio  of  H  to  C02  is  about  2  :  1  for  dextrose  and  lactose. 
In  broth  it  produces  a  general  turbidity  without  film 
formation,  and  the  culture  gives  the  indole  reaction  on 

1  Journ.  Pathol.  and  Bact.,  November  1904. 


384  A  MANUAL  OF  BACTERIOLOGY 

the  addition  of  a  nitrite  in  twenty-four  to  forty-eight 
hours. 

The  fermentation  reactions  are  given  in  the  Table, 
p.  381 .  It  will  be  seen  that  the  B.  coli  is  an  active  fermenter 
of  many  carbohydrates,  alcohols,  and  glucosides,1  e.g. 
glucose,  lactose,  galactose,  mannitol  and  dulcitol,  but  not 
of  adonit.  Cane-sugar  may  or  may  not  be  fermented  ; 
sometimes  only  acid  is  formed,  sometimes  both  acid  and 
gas  are  produced.  To  the  variety  producing  both  acid 
and  gas  from  cane-sugar  Durham  gave  the  name  B.  coli 
communior.  Prescott  and  Winslow  consider  that  the  term 
B.  coli  should  be  applied  only  to  an  organism  that  does  not 
attack  ketonic  sugars.  Neutral  red  in  glucose  broth  is 
changed  to  a  fluorescent  yellow,  and  Houston  describes 
a  typical  B.  coli  as  "  flaginac,"  i.e.  producing  fluorescence 
in  neutral  red  glucose  pep  tone- water  (fl),  acid  and  gas  from 
glucose  (ag),  indole  in  pep  tone- water  (in),  and  acid  and 
curd  in  milk  (ac).  The  colonies  on  Conradi-Drigalski  agar 
are  large  and  red  (see  "  Water  ").  The  B.  coli  does  not 
give  the  Voges-Proskauer  reaction  (p.  389). 

The  differentiation  of  the  B.  coli  from  the  B.  typhosus 
should  present  no  difficulty  if  the  morphology  and  motility 
of  the  organisms  and  their  fermentation  and  agglutination 
reactions  be  compared.  Bacteriologists  usually  make  use 
of  the  following  tests  for  the  differentiation  of  B.  coli  : 
(1)  Morphology,  (2)  motility,  (3)  Gram  staining,  (4)  char- 
acter of  growth  and  colonies  on  gelatin,  (5)  non-lique- 
faction of  gelatin,  (6)  action  on  milk,  (7)  indole  formation, 
(8)  fermentation  of  glucose,  (9)  fermentation  of  lactose 
and  saccharose,  (10)  action  on  neutral  red.  MacConkey 
suggests  that  instead  of  tests  Nos.  4,  6,  7,  8,  and  10,  the 
following  should  be  substituted  :  (a)  fermentation  of 
dulcitol,  but  not  of  adonit  and  inulin  ;  (6)  the  Voges- 
Proskauer  reaction. 

1  Sec  Twort,  Proc.  Roy.  Soc.  Lond.,  B,  vol.  Ixxviii,  p.  329. 


THE  COLON  BACILLUS  385 

Other  media  which  have  been  recommended  for  the  differentia- 
tion of  B.  coli  from  JB.  typhosus  are  the  Proskauer-Capaldi  media 
and  Petruschky's  litinus  whey,  but  are  not  now  much  used. 

The  Proskauer-Capaldi  medium  No.  1  is  an  asparagin-mannitol 
solution  with  certain  salts  ;  medium  No.  2  is  a  peptone-water  - 
mannitol  solution.  Both  solutions  are  carefully  neutralised  and 
tinged  with  litmus. 

If  these  media  be  inoculated  with  B.  typhosus  and  B.  coli  respec- 
tively and  incubated  at  37°  C.  for  twenty-four  hours,  the  following 
changes  will  be  noted  : 

Medium  No.  \.  Medium  No.  2. 

B.  typhosus       No  growth  or  change  Growth  with  strongly 

in  reaction.  acid  reaction. 

B.  coli       .        Growth  with  acid  Growth  with  neutral 

reaction  or  faintly  alkaline 

reaction. 

Petruschky's  litmus  whey  is  prepared  as  follows :  Fresh  milk  is 
warmed  and  the  casein  precipitated  by  the  addition  of  a  minimal 
amount  of  hydrochloric  acid.  It  is  filtered,  and  the  filtrate  of  clear 
whey  is  carefully  neutralised  with  dilute  caustic  soda  solution. 
The  fluid  is  then  steamed  for  two  hours  and  filtered  ;  the  filtrate 
should  be  clear,  colourless,  and  neutral  in  reaction.  Enough  neutral 
litmus  solution  is  then  added  to  render  it  well  coloured,  and  the 
mixture  is  distributed  into  test-tubes  and  sterilised.  This  medium 
is  rendered  slightly  acid  (represented  by  6-10  c.c.  N/ 10  caustic  soda 
per  cent.)  by  B.  typhosus,  very  acid  (40-50  c.c.  ditto)  by  B.  coli. 

The  thermal  death-point  of  the  organism,  according 
to  Weisser  and  Sternberg,  is  60°  C.  with  an  exposure  of 
ten  minutes.  The  B.  coli  will  grow  freely  in  a  slightly 
acid  medium,  and  in  media  containing  as  much  as  0*15 
per  cent,  of  carbolic  acid.  In  this  respect  it  is  a  more 
resistant  organism  than  the  B.  typhosus. 

Chemical  products. — The  acids  produced  are  mainly 
laevo-lactic  acid  with  some  dextro-lactic  acid  from  glucose, 
laevo -lactic  acid  only  from  mannitol ;  also  acetic,  formic 
and  succinic  acids,  and  alcohol.  According  to  Harden, 
B.  coli  attacks  glucose  in  a  characteristic  manner,  each 
molecular  proportion  of  sugar  yielding  half  a  molecular 
proportion  of  acetic  acid  and  of  alcohol,  and  one  molecular 

25 


386  A  MANUAL  OF  BACTERIOLOGY 

proportion  of  lactic  acid,  together  with  a  small  amount  of 
succinic  acid,  and  gaseous  carbonic  acid  and  hydrogen.1 
Nitrates  are  reduced  to  nitrites. 

No  toxin,  or  a  trace  only,  is  formed  in  cultures,  but 
the  dead  bacilli  are  toxic  and  pyogenic,  and  a  toxin  is 
obtained  by  autolysis  of  cultures  or  by  triturating  the 
bacilli  with  liquid  air  (Macfadyen). 

Vaughan,2  by  washing  large  quantities  of  colon  and 
typhoid  bacilli,  extracting  the  bacterial  cells  first  with 
alcohol,  then  with  ether,  and  then  digesting  the  ground 
residue  with  alcohol  containing  2  per  cent.  NaOH,  states 
that  two  constituents  are  obtained,  one  soluble  in  alcohol 
and  toxic,  the  other  insoluble  in  alcohol  and  non-toxic. 
The  latter  confers  a  certain  degree  of  immunity  on  animals 
injected  with  it. 

Pathogenicity. — The  pathogenic  action  and  pathogenicity 
of  the  B.  coli  are  very  varied.  Introduced  into  the  circu- 
lation or  into  the  peritoneal  cavity  in  guinea-pigs  or  rabbits 
it  usually  causes  death  in  from  one  to  three  days  with  a 
general  septicaemia.  Some  varieties  are,  however,  non- 
virulent  to  animals. 

In  man  the  colon  bacillus  is  associated  with  a  number 
of  important  pathological  processes.  It  is  usually  the 
organism  causing  the  peritonitis  which  is  due  to  infection 
from  the  intestine,  as  in  hernia  with  obstruction  or  per- 
foration, in  ulceration  of  the  bowel  and  enteritis,  in  can- 
cerous growths,  and  affections  of  the  appendix,  biliary 
canals,  and  gall-bladder.  The  exudation  in  these  cases 
is  often  characteristic  ;  at  first  it  is  clear  and  greenish, 
it  then  becomes  greenish-yellow,  thin,  semi-opaque  and 
foul-smelling,  and  finally  purulent.  The  colon  bacillus 
may  pass  through  the  intestinal  wall  where  it  has  been 
damaged,  but  not  yet  perforated,  as  in  strangulation. 

1  See  also  Eevis,  Cenlr.  f.  Bakt.  (2t°  Abt.),  xxvi,  1910,  p.  161. 

2  Trans.  XIV  Internal.  Cong.  Hygiene  (Berlin,  1907),  Bd.  iv,  p.  28. 


PATHOGENICITY  OF  COLON  BACILLUS     387 

The  B.  coli  is  a  pyogenic  organism,  and  has  been  met 
with  in  ischio-rectal  abscesses  (probably  the  B.  pyogenes 
fetidus  of  Passet).  Possibly  it  causes  in  some  instances 
the  pneumonia  and  pleurisy  occurring  after  peritonitis, 
for  it  has  been  obtained  from  the  lung  and  pleura  in  these 
conditions,  but  it  must  be  recognised  that  the  B.  coli 
is  a  common  secondary  or  terminal  infection.  B.  coli 
sometimes  induces  puerperal  fever  and  other  forms  of 
septicaemia  and  it  is  a  common  cause  of  cystitis  and  other 
infections  of  the  urinary  tract. 

In  the  Pictou  cattle  disease,  characterised  by  extensive 
hepatic  cirrhosis,  Adami  found  a  minute  diplococcus  or 
short  bacillus.  A  similar  form  was  afterwards  isolated 
by  him  in  hepatic  cirrhosis  in  man.  Miss  Abbot,1  from 
a  study  of  several  such  cases,  came  to  the  conclusion 
that  this  organism  is  a  variety  of  the  B.  coli.  It  has  been 
suggested  that  hepatic  cirrhosis  is  produced  by  poisons 
or  toxins,  e.g.  of  the  B.  coli,  and  that  alcoholism,  the  usual 
cause  assigned,  is  but  an  exciting  or  secondary  agent. 

Anti-serum  and  vaccine. — Attempts  have  been  made  to 
prepare  an  anti-serum  for  B.  coli  infections,  but  they  have 
met  with  little  or  no  success. 

A  vaccine  prepared  by  sterilising  cultures  by  heat  and 
standardising  has  been  used  successfully  in  the  treatment 
of  chronic  B.  coli  infections,  e.g.  cholangitis,  cholecystitis, 
pyelitis,  and  cystitis.  The  B.  coli  vaccine  is  more  toxic 
than  most  vaccines,  and  small  doses  must  therefore  be 
given  (see  p.  221). 


Clinical  Examination 

(1)  The  appearance  and  odour  of  the  pus  are  often  characteristic. 
Smears  of  the  pus  show  small  bacilli,  which  are  decolorised  by 
Gram's  method. 

1  Journ.  Path,  and  Bad.,  vol.  vi,  1900,  No.  3,  p.  315  (Bibliog.). 


388  A  MANUAL  OF  BACTERIOLOGY 

(2)  The  organism  may  be  isolated  by  plating  on  gelatin,  agar, 
litmus  lactose  agar,  Conradi-Drigalski  agar,  or  by  the  use  of  neutral 
red  or  bile -salt  media  (see  "  Water  ").     The  isolated  organism  must 
be  tested  as  to  its  morphology,  motility,  non-Gram  staining,  non- 
liquefaction  of  gelatin,  indole  production,  curdling  of  milk,  and 
fermentation  of  glucose,  lactose,  dulcitol,  mannitol,  etc. 

(3)  An  agglutination  reaction  may  likewise  be  tried,  but  if  nega- 
tive is  of  little  value,  as  there  are  so  many  varieties  of  the  colon 
bacillus,  and  one  variety  may  not  be  agglutinated  by  the  specific 
serum  obtained  with  another  variety.     A  positive  reaction  must 
also  be  carefully  controlled,  as  the  colon  bacilus  is  much  more 
readily  agglutinated  by  normal  serum  than  is  the  typhoid  bacillus. 


Varieties  of  Bacillus  coli 

Organisms  are  frequently  met  with  in  faeces,  manure,  sewage  and 
polluted  water  which  resemble  the  typical  B.  coli  in  many  of  their 
characters,  but  which  differ  from  it  in  certain  particulars.  Thus 
the  colonies  on  gelatin,  instead  of  being  smooth,  may  be  wrinkled  ; 
milk  may  be  but  slowly  curdled  (three  to  eight  days) ;  acid  or  gas 
production,  or  both,  in  sugars  may  be  less  marked  than  usual. 
These  organisms  are  generally  regarded  as  varieties  of  the  B.  coli, 
and  are  perhaps  dervied  from  typical  B.  coli.  There  is,  however, 
little  evidence  that  B.  coli  can  be  transformed  into  such  varieties, 
or  that  these  varieties  can  be  reconverted  into  typical  B.  coli  ; 
Revis  (loc.  cit.)  has  produced  considerable  alterations  of  fermentive 
power,  and  in  the  characters  of  the  colonies  of  certain  coliform 
organisms. 

Organisms  that  have  been  Regarded  as 
Variants  of  B.  coli 

A  number  of  organisms  have  been  regarded  as  being  varieties  of 
the  B.  coli  (consult  Table  of  fermentation  reactions,  p.  381). 

(1)  Bacillus  cavicida  (Brieger). — This  resembles  B.  coli  in  most 
of  its  characters,  but  was  stated  to  be  non-motile.     MacConkey  says 
it  is  motile. 

(2)  Bacillus  neapolitanus  (Emmerich). — Isolated  from  the  bowel 
in  cases  of  cholera.     It  differs  from  B.  coli  in  not  being  motile,  and 
in  fermenting  cane  sugar. 

(3)  Gas-forming  bacilli  of  Laser  and  Gartner.1 

1  Oentr.f.  BaJct  (lte  Abt.),  xiii,  1893,  p.  217  ;   xv,  1894,  pp.  1,  276. 


FLIES  AS  CARRIERS  OF  INFECTION        389 

(4)  Aerobic  bacillus  of  malignant  oedema  (Klein). 

(5)  Bacillus  lactis  aerogenes  of  Escherich. — Found  in  the  intestine 
of  nurslings  and  in  milk.     Much  like  B.  coli,  but  is  non-motile. 
It  differs  from  B.  coli  by  not  fermenting  dulcitol,  by  fermenting 
saccharose  and  adonit,  and  by  giving  the  Voges-Proskauer  reaction 
(see  Table,  p.  381).     According  to  Harden  and  Walpole,1  its  action 
on  glucose  differs  from  that  of  B.  coli,  more  alcohol  being  produced 
and  formed  at  the  expense  of  that  part  of  the  molecule  of  the  sugar 
which  in  the  B.  coli  fermentation  yields  acetic  and  lactic  acids. 

The  Voges-Proskauer  reaction  is  obtained  by  growing  the 
organism  in  2  per  cent,  glucose  broth  in  a  fermentation  tube  (Fig.  17, 
p.  84)  for  three  days  and  adding  some  strong  caustic  potash  solu- 
tion ;  on  standing  exposed  to  the  air  a  pink  colour  develops. 
According  to  Harden  and  Walpole  2  the  reaction  is  probably  due 
to  acetylmethyl-carbinol,  which  in  the  presence  of  air  and  potash 
is  oxidised  into  diacetyl,  which  then  reacts  with  some  constituent 
of  the  peptone  in  the  medium,  giving  the  pink  colour. 

The  B.  lactis  aerogenes  (which  may  be  classed  among  the  capsu- 
lated  bacilli,  see  p.  258)  is  occasionally  pathogenic,  causing  peri- 
tonitis. 3  In  these  circumstances,  it  is  capsulated,  but  the  capsule  is 
difficult  to  stain.  It  seems  probable  that  the  B.  capsulatus  of 
Pfeiffer  is  identical  with  this  organism. 

(6)  B.  cloacae,  (Jordan). — Met  with  in  sewage.     In  general  char- 
acters it  much  resembles  B.  coli,  but  produces  more  gas  (75  per  cent.) 
from  glucose  and  liquefies  gelatin  in  four  or  five  to  thirty  days.  Like 
B.  lactis  aerogenes,  saccharose  is  always  fermented  and  the  Voges- 
Proskauer  reaction  is  positive,  but  neither  dulcitol  nor  adonit  is 
fermented.     (See  Table,  p.  381.) 


Flies  as  Carriers  of  Infection 

Flies  and  other  "insects  "  may  convey  infection  (1)  by  acting  as 
"  porters  "  and  infecting  food,  etc.,  (2)  by  direct  inoculation,  (3)  by 
inoculation  after  a  cycle  of  development — in  which  case  the  carrier 
is  more  or  less  specific  ;  e.g.  anopheline  mosquitoes  in  malaria.  In 
the  first  method  the  organisms  are  generally  bacteria,  occasionally 
ova  of  worms  ;  in  the  second,  bacteria  or  protozoa  ;  in  the  third, 
invariably  protozoa,  filaria,  etc.,  i.e.  animal  organisms. 

1  Journ  of  Hygiene,  vol.  v,  1905,  p.  488  ;   Proc.  Roy.  Soc.  Lond.,  B, 
vol.  Ixxvii,  1906,  p.  399. 

2  Proc.  Roy.  Soc.  Lond.,  B,  vol.  Jxxvii,  1906,  p.  399. 

3  See  Churchman,  Johns  Hopkins  Hosp.  Bull.,  vol.  xxii,  1911,  p.  116. 


390  A  MANUAL  OF  BACTERIOLOGY 

The  ordinary  domestic  fly,  the  blue-bottle  and  other  similar  flies 
(of  which  there  are  many)  have  no  biting  proboscis,  but  undoubtedly 
directly  convey  infection  to  food,  etc.,  by  carrying  organisms  upon 
various  parts  of  their  body,  or  by  the  organisms  passing  through 
the  digestive  tract  and  infecting  the  food  with  the  faeces.  In  tin's 
way,  typhoid,  bacillary  dysentery,  B.  enteritidis,  summer  diarrhoea, 
cholera,  and  possibly  anthrax,  and  also  the  ova  of  certain  worms, 
may  be  conveyed. 

The  ordinary  house-fly  breeds  in  dung  and  garbage  containing 
dung,  and  it  has  a  possible  range  of  flight  of  about  a  mile.  The 
house-fly  experimentally  infected  remains  grossly  infected  for  at 
least  three  days,  and  a  smaller  degree  of  infection  persists  for  ten 
days  or  even  longer.  1 

1  Sec  Reports  to  the  Loc.  Gov.  Board  on  Flies  as  Carriers  of  Infection, 
Nos.  1-4,  1910  and  1911.  Martin,  Brit.  Med.  Jovrn.,  1913,  I.,  p.  1. 


CHAPTER  XI 

BUBONIC  PLAGUE— CHICKEN  CHOLERA- 
MOUSE  SEPTICAEMIA 

Bubonic  Plague 

PLAGUE  was  epidemic  throughout  Europe  during  the 
Middle  Ages  ;  in  England  in  the  fourteenth  century  it 
appeared  as  the  Black  Death,  and  in  the  seventeenth 
century  as  the  Great  Plague  of  London,  while  numerous 
other  lesser  visitations  have  been  recorded.  For  some 
years  plague  has  been  practically  pandemic.  The  disease 
seems  always  to  have  been  endemic  in  certain  centres, 
e.g.  in  Asia  Minor,  on  the  Persian  Gulf,  in  Yunnan,  in 
Uganda,  etc.  A  characteristic  of  plague  is  the  manner 
in  which  it  appears  and  remains  prevalent  for  a  time  in 
a  district  and  then  disappears,  to  reappear  again  after  a 
considerable  interval ;  this  has  happened  not  only  in 
Europe,  but  also  in  Persia,  Syria,  India,  and  China. 

Three  principal  types  of  the  disease  are  recognised,  the 
bubonic  in  which  the  femoral  (rarely  the  inguinal),  axillary 
and  other  glands  become  enlarged  (whence  the  disease 
derives  its  name),  the  septicaemic,  and  the  pneumonic. 
In  India  the  disease  has  been  mainly  bubonic  (70  per  cent, 
of  the  cases).  Occasionally  the  majority  of  the  cases  are 
pneumonic,  as  was  the  case  in  Accra,  in  China  in  1910-11, 
and  in  the  small  outbreak  in  Suffolk  in  1910.  Septicsemic 
cases  are  the  exception,  but  any  form  tends  to  become 
septicsemic  on  the  approach  of  death. 

391 


392  A  MANUAL  OF  BACTERIOLOGY 

At  the  commencement  and  at  the  end  of  an  epidemic 
the  disease  may  assume  an  extremely  mild  type,  the 
so-called  "  pestis  minor." 

Bacilli  were  first  observed  in  this  disease  in  the  blood, 
buboes,  and  organs  by  Kitasato  in  1894.  In  the  same 
year  (1894)  Yersin  investigated  the  outbreak  of  bubonic 
plague  at  Hong  Kong,  and  described  the  bacillus  met 


FIG.  43. — Smear  preparation  from  spleen  of  inoculated 
guinea-pig,     x  1000. 

with  in  the  buboes  and  its  cultural  and  pathogenic  properties 
very  fully.  This  organism  is  known  as  the  Bacillus  pestis. 
Morphology. — The  B.  pestis  belongs  to  the  group  of 
hsemorrhagic  septicsemic  bacilli  (chicken  cholera,  rabbit 
and  ferret  septicaemia,  swine  plague,  etc.,  see  p.  404), 
and  is  a  markedly  pleomorphic  organism.  In  the  animal 
body  it  occurs  for  the  most  part  as  a  short,  plump,  non- 
sporing  rod,  measuring  2-3  /m  by  1-2  /u.,  but  longer  forms 
may  be  seen  here  and  there  measuring  as  much  as  5  ju, 
(Fig.  43).  Polar  staining  is  a  marked  feature  (Plate  XIV. 
a  and  6),  and  swollen  involution  forms  occasionally  occur. 


PLATE  XIV. 


a.  Bacillus  pestis.     Smear  preparation  from  a  bubo,      x  1000. 


b.  Bacillus  pestis.     Smear  preparation  of  sputum,      x  1000. 


THE  PLAGUE  BACILLUS  393 

The  typical  form  of  the  organism,  the  bi-polar  staining, 
short,  stumpy  bacillus,  is  met  with  in  smears  from  the 
buboes,  in  the  sputum  in  the  pneumonic  form,  and  in  the 
blood  in  the  septicsemic  variety,  but  only  in  the  earlier 
stages  of  the  disease.  Later  the  typical  forms  tend  to 
disappear,  their  place  being  taken  by  a  few  large,  rounded, 
ovoid,  or  pear-shaped  involution  forms.  Under  cultiva- 
tion the  bacilli  in  young  cultures  (twenty-four  to  forty- 
eight  hours)  are  so  short  as  to  be  almost  coccoid  or  slightly 
ovoid  ;  on  agar  their  size  is  about  the  same  as  that  in  the 
animal  body,  on  gelatin  they  are  somewhat  smaller,  but 
a  few  well-marked  rods  and  even  threads  are  always  present. 
In  older  cultures,  rod,  thread  and  involution  forms  occur 
more  numerously ;  on  agar  containing  2-3  per  cent,  of 
salt  the  latter  are  swollen  and  yeast-like. 

In  broth  chains  of  slightly  ovoid  organisms  occur 
resembling  streptococci  (Plate  XV.  a). 

The  organism  is  non-sporing  and  non-motile,  although 
Gordon  described  the  presence  of  one  or  two  fine  spiral 
terminal  flagella  (others  have  not  found  flagella). 

Sometimes  in  hanging- drop  cultivations  a  capsule  is 
apparently  present,  but  the  writer  has  failed  to  verify  this 
by  staining  methods. 

The  B.  pestis  stains  well  with  Loffler's  blue  and  anilin- 
gentian  violet,  polar-staining  being  a  marked  feature, 
especially  in  smear  preparations.  It  does  not  stain  by 
Gram's  method.  With  old  laboratory  strains  polar  stain- 
ing may  be  completely  absent,  but  in  such  cases  may 
sometimes  be  obtained  by  first  treating  the  preparations 
with  alcohol  or  by  the  Gram  method,  and  subsequently 
staining  with  Loffler's  blue  or  weak  gentian  violet.  Sections 
are  best  stained  with  carbol  methylene  or  thionine  blue. 

Cultural  characters. — The  B.  pestis  is  aerobic  and  facul- 
tatively anaerobic.  On  blood-serum  it  forms  moist, 
smooth,  shining,  cream-coloured  colonies  or  growths, 


394  A  MANUAL  OF  BACTERIOLOGY 

slightly  raised  above  the  surrounding  medium.  The 
blood-serum  is  not  liquefied. 

On  agar  the  colonies  are  raised,  round  and  cream- 
coloured,  finely  granular,  denser  at  the  centre  than  at 
the  margins,  which  are  regular.  Size  0-25  to  0-5  mm.  in 
two  days  at  37°  C. 

On  surface  agar  the  B.  pestis  forms  a  thick,  opaque, 
moist,  smooth,  cream-coloured  growth,  the  margins  of 
which  are  usually  markedly  crenated ;  the  growth  is 
very  sticky  and  tenacious.  Haffkine  states  that  when 
grown  on  dry  agar  (agar  which  has  been  kept  in  the  warm 
incubator  for  two  to  three  weeks)  and  viewed  from  behind 
the  growth  has  an  appearance  like  that  given  by  the 
back  of  a  mirror — i.e.  a  dull,  silvery  appearance. 

On  a  salt  agar  (2-5-3-5  per  cent,  of  sodium  chloride) 
Hankin  describes  the  development  of  remarkable  spherical 
or  pear-shaped  involution  forms. 

On  gelatin  the  colonies  are  whitish,  filmy,  finely  granular 
with  regular  margins.  Size,  0-1  to  0-25  mm.  in  five  days 
at  22°  C. 

On  surface  gelatin  the  organism  forms  a  thin,  white, 
granular  growth,  with  slightly  irregular  surface  and  margins, 
and  nearly  confined  to  the  inoculation  track  (Fig.  44). 
The  growth  does  not  penetrate  into  the  medium,  nor  does 
it  render  it  cloudy.  The  growth  is  very  adherent. 

In  a  stab  gelatin  culture  a  delicate  whitish,  finely  granular 
growth  develops  to  the  end  of  the  stab,  with  little  tendency 
to  spread  from  the  needle  track.  The  gelatin  is  not 
liquefied.  Both  in  agar  and  gelatin  cultures  fresh  punctate 
growths  sometimes  develop  in  the  original  growth,  simu- 
lating a  contamination.  No  growth  occurs  on  ordinary 
potato,  and  milk  is  not  coagulated. 

In  broth  the  growth  is  somewhat  characteristic.  For 
two  or  three  days  the  broth  remains  perfectly  clear,  but 
a  flocculent  growth  forms  and  gradually  increases  in 


PLATE  XV. 


a.  Bacillus  pestis.     Film  preparation  from  a  72-hours'  broth 
culture,      x  1000. 


6.  Chicken  cholera.     Film  preparation  of  blood  of  fowl. 
X  1000. 


THE  PLAGUE  BACILLUS 


395 


amount  on  the  bottom  and  sometimes  upon  the  sides  of 
the  tube.  After  some  days  the  broth  may  become  a  little 
cloudy.  A  delicate  flocculent  film  develops  if  the  tube 
be  kept  absolutely  at  rest.  In  broth  to  which  a  little 
butter-fat  or  ghee  has  been  added  little  islands  of  growth 
appear  on  the  surface,  and  from 
these  flocculent  tapering  depen- 
dent growths  form  in  about  a 
week,  provided  the  tubes  or 
flasks  be  kept  absolutely  at  rest, 
the  bulk  of  the  broth  remaining 
clear.  This  is  the  stalactite 
growth  of  Haffkine,  and  is  very 
characteristic  (B.  pseudo-tuber- 
culosis also  gives  it).  Broth 
cultures  reduce  a  weak  solution 
of  methylene  blue. 

With  sulphuric  acid  alone  a 
feeble  indole  reaction  can  be 
obtained  with  week- old  broth 
cultures.  With  sulphuric  acid 
and  a  nitrite  a  well-marked 
indole  reaction  can  be  obtained 
under  the  same  conditions. 

The  fermentation  reactions  of 
the  B.  pestis,  which  MacConkey 
has  pointed  out  are  practically  identical  with  those  by  the 
B.  pseudo-tuberculosis,  are  as  follows  :  Acid  production, 
but  no  gas,  in  glucose,  laevulose,  galactose,  maltose, 
mannitol,  and  dextrin,  no  change  in  lactose,  cane-sugar, 
and  dulcitol. 

Action  of  antiseptics,  etc. — The  plague  bacillus  is  readily 
destroyed  by  antiseptics  ;  a  1  :  1000  corrosive  sublimate 
or  1  :  100  chloride  of  lime  solution  being  efficient.  An 
acid  solution  of  corrosive  sublimate  is  preferable,  and  for 


FIG.  44. — Plague,  surface  cul- 
ture on  gelatin  four  days 
old. 


396  A  MANUAL  OF  BACTERIOLOGY 

the  practical  disinfection  of  native  houses  a  1  :  250  solution 
of  sulphuric  acid  may  be  employed.  A  temperature  of 
65°  C.  kills  the  organism  in  about  fifteen  minutes.  Desic- 
cation over  sulphuric  acid  at  30°  C.  is  also  rapidly  fatal. 

Vitality  and  virulence  of  cultures. — Cultures  retain  their 
vitality  for  at  least  a  month.  As  regards  virulence,  the 
organism  varies  much  according  to  the  source  from  which 
it  is  obtained.  Under  cultivation  it  gradually  loses  its 
virulence  unless  subcultured  in  the  following  manner  : 
The  cultures  are  made  every  week  on  surface  agar,  are 
placed  in  the  blood-heat  incubator  for  twenty-four  hours, 
and  are  then  removed  and  kept  at  room  temperature.  If 
inoculated  into  animals  the  virulence  may  be  heightened 
for  a  particular  species  by  successive  passages,  but  in 
so  doing  is  diminished  for  other  species. 

Pathogenic  action. — In  addition  to  man,  the  following 
animals  are  liable  to  contract  plague  under  natural  con- 
ditions— the  monkey,  cat,  rat,  mouse,  squirrel,  ground 
squirrel,  ferret,  bandicoot,  and  marmot.  The  guinea- 
pig  and  rabbit  are  also  susceptible  to  inoculation.  The 
horse,  cattle,  sheep  and  goat  are  relatively  insusceptible, 
though  Simpson  *  stated  that  calves  and  poultry  may  be 
infected  by  feeding,  and  suffer  from  a  chronic  form  of  the 
disease  (this  observation  of  Simpson's  has  not  been  con- 
firmed by  other  workers).  Birds  are  not  easily  susceptible, 
and  vultures  feeding  on  the  corpses  of  the  plague- stricken 
do  not  seem  to  contract  the  disease.  The  mouse,  rat,  and 
guinea-pig  are  the  animals  chiefly  used  for  experimental 
purposes  in  the  laboratory ;  the  first  two  are  highly 
susceptible,  a  simple  prick  in  the  thigh  with  an  infected 
needle  being  sufficient  to  induce  the  disease. 

A  guinea-pig  inoculated  with  plague  material  or  with 
a  pure  cultivation  usually  dies  in  from  two  to  seven  days, 
the  symptoms  being  sluggishness  and  loss  of  appetite, 

1  Report  on  the  Plague  in  Hong  Kong. 


PATHOGENICITY  OF  PLAGUE  BACILLUS    397 

sometimes  a  discharge  from  the  eyes,  and  towards  the 
end,  staring  coat  and  perhaps  convulsive  and  paralytic 
attacks.  The  post-mortem  appearances  are  extensive 
haemorrhagic  oedema  at  the  seat  of  inoculation,  enlarge- 
ment and  congestion  of  the  spleen,  and  enlargement  of, 
and  hemorrhages  into,  the  inguinal  and  axillary  lymphatic 
glands.  If  the  animal  live  six  or  seven  days,  the  glands 
may  be  as  large  as  small  nuts  (see  some  admirable  prepara- 
tions in  the  College  of  Surgeons  Museum).  The  spleen 


FIG.  45. — Spleen  of  guinea-pig  inoculated  with  plague. 
(Nat.  size.) 

may  be  enormous,  six  times  its  natural  size,  and  studded 
with  small  yellowish  nodules  resembling  miliary  tubercles, 
consisting  of  necrotic  areas  with  masses  of  bacilli  (Fig.  45) ; 
the  lungs  also  may  be  more  or  less  inflamed,  and  contain 
small  and  large  necrotic  foci.  The  bacilli  are  extremely 
numerous  at  the  seat  of  inoculation,  in  the  glands,  and 
in  the  spleen,  less  so  in  the  peritoneal  fluid,  liver,  and 
blood  ;  if  the  death  of  the  animal  is  delayed  the  exudation 
in  the  bronchi  may  contain  considerable  numbers.  Some 
bacilli  may  generally  be  found  in  the  duodenum,  trachea, 
and  larynx.  Mice  usually  die  in  from  two  to  three  days, 
and  rats  in  from  three  to  seven  days  after  inoculation.  In 
rats  and  mice  the  post-mortem  appearances  are  similar 
to  those  in  the  guinea-pig.  A  very  small  dose  of  a  pure 
culture  may  fail  to  kill  an  inoculated  animal.  Rabbits 
are  much  less  susceptible  to  plague  than  guinea-pigs,  and 
may  be  injected  with  considerable  doses  of  living  cultures 
without  showing  marked  illness.  Rats  can  be  infected 


398  A  MANUAL  OF  BACTERIOLOGY 

by  feeding  on  the  corpses  or  carcases  of  men  or  animals 
dead  from  the  disease. 

In  man  the  bacilli  are  found  in  large  numbers  in  the 
fluid  in  the  buboes,  either  alone  or  mixed  with  streptococci 
or  micrococci,  and  in  the  sputum  in  the  pneumonic  form. 
They  are  not  usually  found  in  any  number  in  the  blood 
except  in  the  septicaemic  variety,  or  shortly  before  death, 
and  in  stained  preparations  appear  as  short  plump  bacilli, 
often  in  pairs,  with  polar  staining  and  unstained  centres 
(Plate  XIV.  a  and  6).  If  the  organisms  are  found  to  be 
free  and  numerous  in  the  buboes  the  prognosis  tends  to 
be  grave,  but  if  they  are  largely  present  within  the 
phagocytic  polymorphonuclear  leucocytes  the  prog- 
nosis is  better  and  the  disease  will  probably  remain 
localised.  • 

Toxins. — The  plague  bacillus  forms  but  little  toxin,  the 
minimal  fatal  dose  of  the  most  active  filtered  broth  culture 
for  a  mouse  being  about  0-02  c.c.  In  order  to  prepare  a 
vaccine  or  an  anti-serum  it  is  necessary,  therefore,  to 
employ  unfiltered  cultures  -4.e.  the  microbes  themselves. 

Macfadyen  obtained  an  endotoxin  by  triturating  the 
bacilli  frozen  with  liquid  air. 

Vaccines  and  immunity. — Of  the  plague  vaccines,  that 
of  Haffldne,  the  Haffldne  prophylactic,  is  the  best  known, 
and  has  been  extensively  employed.  It  consists  essen- 
tially of  a  four  to  six  weeks  old  butter- fat  broth  culture 
of  the  plague  bacillus,  killed  by  heating  to  65°  C.  for  an 
hour,  with  a  small  addition  of  antiseptic.  As  to  the 
value  of  Hafikine's  prophylactic  a  mass  of  figures  is 
available.  By  its  use  both  the  incidence  of,  and  mortality 
from,  plague  are  markedly  diminished.  Wilkinson  col- 
lected the  following  data  of  the  efficiency  of  the  vaccine  : 
Among  the  inoculated  the  case  incidence  was  1*8  and  the 
case  mortality  23-9  per  cent. ;  among  the  uninoculated 
the  figures  were  7-7  and  60-1  respectively.  The  immunising 


PLAGUE  VACCINES  399 

products  seem  to  be  mainly  intracellular,  but  the  broth 
itself  is  not  without  action. 

Other  vaccines  have  also  been  devised.  Lustig  and  Galeotti 
prepared  one  by  digesting  the  growth  from  agar  cultures  with  1  per 
cent,  caustic  soda  solution,  filtering  through  paper,  and  precipi- 
tating with  very  dilute  acetic  or  hydrochloric  acid,  or  by  saturation 
with  ammonium  sulphate.  The  precipitate  is  dissolved  in  a  0-5  per 
cent,  solution  of  sodium  carbonate,  and  filtered  through  a  Chamber- 
land  filter  ;  this  forms  the  vaccine  fluid.  Calmette  prepared  a 
vaccine  by  emulsifying  an  agar  growth  in  water,  well  washing  the 
organisms  with  sterile  water  to  remove  adherent  toxin,  emulsifying 
again  in  sterile  water,  heating  to  70°  C.  for  an  hour,  and  finally 
drying  in  vacuo.  The  dry  substance  can  be  kept  for  a  considerable 
time  without  change.  For  use  1-2  mgrm.  are  emulsified  in  2-3  c.c. 
of  sterile  salt  solution  and  injected. 

Yersin  proposed  vaccinating  with  living  culture  of  feeble  viru- 
lence, which  has  been  done  by  Strong  in  Manila.  Though  such  a 
method  might  be  used  in  a  plague -stricken  district,  it  is  obviously 
one  that  can  be  used  only  with  the  greatest  caution. 

Klein  1  has  prepared  a  prophylactic  by  drying  the  organs  of  a 
guinea-pig  dead  of  plague  for  three  days  at  46°  C.,  rubbing  the 
material  to  a  powder,  and  further  drying  at  37°  C.  for  three  days. 
Of  this  dry  powder  15-16  mgrm.  protected  a  rat,  and  25  mgrm.  a 
monkey. 

With  reference  to  experimental  immunity  and  protection  in 
plague,  Klein  2  found  that  a  guinea-pig  which  had  been  three  times 
injected  with  an  amount  of  living  culture  insufficient  to  kill  was 
still  capable  of  being  infected  ;  that  the  blood  of  a  guinea-pig  which 
had  twice  passed  through  an  attack  of  plague  did  not  contain  an 
appreciable  amount  of  germicidal  substances  ;  and  that  the  im- 
munisation of  guinea-pigs  by  sterilised  cultures  is  an  extremely 
slow  and  difficult  process.  Calmette  also  found  that  the  guinea-pig 
was  extremely  difficult  to  immunise. 

Calmette,  from  laboratory  experiments,  surmised  that  protection 
with  a  vaccine  is  not  attained  for  some  days,  and  that  in  the  interval 
susceptibility  to  infection  is  increased.  These  observations  are  not 
borne  out  in  practice,  for  Bannerman  3  found  that  so  far  from  there 
being  an  increase  in  mortality  among  those  who  have  been  inocu- 
lated and  who  develop  plague  within  ten  days  of  inoculation  the 

1  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1905-06. 

2  Ibid.  1896-97,  App.  B.,  p.  2. 

3  Centralbl.  f.  Bakt-  /lte  Abt.).  Bd.  xxix,  p.  873  (Bibliog.). 


400  A  MANUAL  OF  BACTERIOLOGY 

reverse  is  the  case,  and  that  in  a  small  community  where  the 
population  had  been  partly  vaccinated  and  partly  not  vaccinated, 
the  incidence  of  plague  during  the  week  following  vaccination  was 
less  among  the  vaccinated  than  among  the  unvaccinated,  pointing 
to  the  rapid  production  of  protection. 

Anti-plague  serum. — This  is  prepared  by  growing  the 
B.  pestis  on  the  surface  of  agar  in  plate  bottles,  washing 
off  and  emulsifying  the  growth,  and  for  the  earlier  injec- 
tions the  emulsion  is  heated  to  65°  C.  for  one  hour,  and 
the  commencing  dose  is  ^  part  of  a  flask.  The  injections 
are  given  intravenously  at  intervals  of  a  week.  At  the 
end  of  three  months  the  bactericidal  power  of  the  blood 
will  have  become  very  marked,  and  living  cultures  are 
then  injected  for  a  further  period  of  about  three  months 
until  a  whole  flask- culture  is  given  at  a  dose.  An  interval 
of  a  fortnight  is  allowed  to  elapse  between  the  last  dose 
and  the  bleeding  of  the  animal.  The  serum  is  tested 
upon  mice. 

The  anti-plague  serum,  which  is  mainly  anti-microbic, 
is  not  very  potent,  and  to  be  of  service  large  amounts  and 
early  treatment  are  essential.1 

Epidemiology. — The  mode  of  infection  in  man  has  been 
a  matter  of  controversy.  The  pneumonic  form  arises 
generally  from  aerial  infection  by  the  respiratory  tract. 
It  is  extremely  fatal  and  infectious,  while  the  bubonic  and 
septicaemic  varieties  are  hardly  ever  contagious.  Although 
a  gastric  and  intestinal  form  of  the  disease  has  been 
described,  and  there  is  evidence  to  show  that  food  or  drink 
may  be  the  vehicle  of  infection,  this  must  be  a  rare  mode 
of  infection.  Yersin  claimed  to  have  isolated  the  bacillus 
from  the  dust  and  earth  of  a  native  dwelling,  and  Hankin 
from  the  brackish  water  in  a  field.  The  observations  of 
Hankin  and  others  indicate,  however,  that  contagion  is 
likely  to  occur  only  from  immediate  contact  with  man  or 

1  See  Hewlett's  Serum  Therapy,  1910. 


TRANSMISSION  OF  PLAGUE  401 

animals,   or  their  excretions,   infected  with  plague,   and 
not  from  a  saprophytic  form  of  the  organism. 

Certain  animals,  especially  the  rat  (Mus  rattus  and  Mus 
decumanus),  are  important  agents  in  spreading  the  disease. 
The  association  of  sickness  and  of  death  among  rats 
with  an  epidemic  of  plague  has  been  established  by  a 
number  of  observations,  and  in  some  instances  the  epizootic 
among  rats  has  been  definitely  shown  to  precede  the 
epidemic  in  man.  The  epidemics  at  Sydney  are  perhaps 
the  most  striking  instances  of  rat-borne  plague  ;  discussing 
the  first  one  Tidswell  says  :  "  The  one  clear  fact  in  our 
epidemic  was  that  human  beings  were  not  becoming  infected 
from  one  another."  In  the  first  epidemic  the  mode  of 
introduction  of  the  disease  was  never  traced  to  any  human 
source.  During  an  epidemic  rats  may  be  found  in 
all  stages  of  illness  and  plague  bacilli  can  be  found  in 
large  numbers  in  their  carcases.  In  the  various  epidemics 
at  Sydney,  cases  of  plague  first  occurred  among  the  rats 
and  mice,  followed  after  an  interval  of  days  or  weeks  by 
human  cases.  Other  animals  may  also  occasionally  be 
the  means  of  disseminating  the  disease.  The  experiments 
of  the  Advisory  Committee  on  Plague  Investigation  in 
India  have  conclusively  shown  the  important  part  played 
by  rats  in  the  dissemination  of  the  disease,  though  the 
origin  of  the  primary  infection  in  rats  is  doubtful.  They 
may  possibly  become  infected  from  the  dust  of  earthen 
floors  of  the  native  houses  soiled  with  excreta  or  discharges 
of  plague  patients,  or  from  their  clothing,  poultices  or 
dressings,  but  the  readiest  method  is  probably  by  feeding 
on  the  dead.  Once  the  epizootic  has  started,  further 
infection  is  simple  ;  rats  fight,  and  so  may  directly  inocu- 
late one  another ;  the  sick  rats  may  soil  grain  or  other 
food-stuffs,  and  the  dead  rats  are  eaten  by  their  fellows. 
Moreover,  parasitic  insects,  especially  fleas,  undoubtedly 
may  transmit  the  disease  from  one  animal  to  another. 

26 


402  A  MANUAL  OF  BACTERIOLOGY 

Thus  it  is  found  that  if  guinea-pigs  be  placed  in  a  plague- 
infected  compound,  many  of  the  animals  contract  plague  ; 
but  if  the  animals  be  placed  in  cages  of  wire-gauze,  the 
mesh  of  which  is  small  enough  to  prevent  access  of  fleas, 
the  animals  do  not  contract  plague.  The  transmission 
of  the  disease  from  rats  to  man  is  similarly  due  to  trans- 
mission by  fleas  (except  in  the  pneumonic  forms  in  which 
infection  is  direct  from  the  sick  to  the  healthy).  The  great 
majority  of  rat  fleas  are  Xenopsylla  cheopis,  Ceratophyllus 
fasciatus,  Cer.  anisus,  Ctenopsylla  musculi,  and  Ctenoph- 
ihalmus  agyrtes,  of  which  the  first  is  most  prevalent  in  the 
tropics  and  subtropical  regions,  the  second  in  cooler  regions 
and  in  this  country.1  Walker  2  has  found  that  bed-bugs 
may  occasionally  transmit  plague.  The  bacilli  multiply 
in  some  of  the  fleas  to  such  an  extent  as  to  occlude  the 
entrance  to  the  stomach.  Such  fleas  will  still  bite,  but 
on  ceasing  to  suck,  some  of  the  blood  with  numerous 
bacilli  in  it  regurgitates  into  the  wound  and  thus  infects.3 
The  seasonal  prevalence  of  plague  coincides  with  the 
prevalence  of  rat-fleas.  The  manner  in  which  the  periods 
in  the  year  when  human  plague  does  not  occur  are  bridged 
over  is  unknown.  In  such  periods  rats  suffering  from 
plague  have  been  found,  but  these  are  regarded  as  having 
a  retrogressive  form  of  the  disease  rather  than  a  chronic 
infection.  The  destruction  of  rats,  either  by  trapping, 
poisoning,  or  asphyxiating,  or  by  the  use  of  the  Danysz 
rat  virus  (see  p.  373),  is,  therefore,  one  of  the  means  to  be 
adopted  in  fighting  the  disease.  The  extermination  of 
rats  seems  quite  impossible,  but  by  rat  destruction  there 
is  a  likelihood  of  destroying  infected  animals  and  the 
subsequent  development  of  a  healthy  race.  On  the  other 
hand,  objection  has  been  taken  to  rat-destruction,  it  being 

1  See  Chick  and  Martin,  Journ.  of  Hygiene,  vol.  xi,  1911,  p.  122. 

2  Ind.  Med.  Gaz.,  May  1910. 

37Bacot    and    Martin,    Journ.  of  Hygiene,  XIII,  Plague  Supp.  Ill 
914,  p.  423. 


DIAGNOSIS  OF  PLAGUE  403 

surmised  that  if  the  epizootic  be  allowed  to  proceed,  the 
susceptible  rats  will  be  exterminated  and  a  race  of  rats 
relatively  insusceptible  to  plague  will  ultimately  be 
established. 

On  Plague,  see  Simpson,  Treatise  on  Plague  (Cambridge  Univer- 
sity Press) ;  Klein,  Bacteriology  of  Oriental  Plague  ;  "  Reports  on 
Plague  Investigations  in  India,"  Journ.  of  Hygiene  (extra  numbers), 
vols.  vi-xiv. 

Clinical  Examination 

If  it  cannot  be  examined  immediately,  plague  material  may  be 
placed  in  a  solution  containing  glycerin  20  c.c.,  distilled  water 
80  c.c.,  calcium  carbonate  2  grm.  The  bacilli  retain  their  vitality 
and  virulence  in  this  for  thirteen  days  (Albrecht-Ghon  method). 

(1)  Withdraw  a  little  of  the  fluid  from  the  bubo  by  means  of  an 
antitoxin  syringe.     Make   smears   and  stain  with   methylene   or 
thionine  blue.     Search  for  short  plump  bacilli,  often  in  pairs,  with 
polar  staining  and  unstained  centres.     They  are  not  stained  by 
Gram's  method. 

N.B. — There  may  be  a  mixture  of  organisms  in  the  buboes. 

(2)  Make  agar  plates  and  broth  cultures.     Incubate  the  cultures 
at  25°-27°  C.,  not  at  37°  C.     From  colonies  on  the  agar  plates  the 
organism  may  be  isolated  and  its  cultural  and  pathogenic  characters 
ascertained.     The   appearance   of  the   broth  cultures,  if  charac- 
teristic, would  be  very  suggestive  of  plague,  but  if  uniform  turbidity 
develops  this  may  be  due  to  contaminating  organisms,  e.g.  micrococci. 

(3)  Inoculate  mice,  rats,  or  guinea-pigs  subcutaneously  with  the 
fluid  or  with  the  culture.     Some  of  the  animals  should  be  inoculated 
by  the  cutaneous  method — rubbing  a  little  of  the  material  on  the 
shaved  abdomen,  and  also  as  in  (4).     Inoculation  of  rats  serves  to 
distinguish  the  B.  pseudo-tuberculosis  from  the  B.  pestis.     If  the 
animals  die,  investigate  for  the  Bacillus  pestis  by  staining  and 
culture  methods. 

(4)  In   the   pneumonic   form,  dilute   the   sputum  with   a  little 
boiled   water,    inoculate    several  agar   tubes,    and    incubate    at 
25°-27°  C.     Examine    in    two   to   three  days.      Also   daub   the 
nostrils  of  a  guinea-pig  or  rat  with  a  brush  or  pledget  of  wool 
dipped   in   the    diluted  sputum,  avoiding  wounding  the  mucous 
membrane.     Smears  of  the  sputum  may  also  be  made,  stained,  and 
examined.     Gram's  method  will  distinguish  the  B.  pestis  from  the 
Streptococcus  pneumonice  ;  the  latter  stains  well  by  Gra.m, 


404  A  MANUAL  OF  BACTERIOLOGY 

(5)  Agglutination  reaction. — The  Indian  Plague  Commissioners 
state  that  in  their  opinion  no  practical  value  attaches  to  the  method 
of  serum  diagnosis  in  plague,  but  a  modified  method  is  considered 
by  Dunbar  x  to  be  of  considerable  value.  The  method  is  carried 
out  as  follows : 

A  small  quantity  of  peptone  solution,  inoculated  with  the  tissue 
juice  from  the  suspected  organ,  is  mixed  with  an  equal  quantity 
of  plague-serum  of  such  a  strength  that  the  dilution  reduces  it  to 
1  :  200  (approximately).  A  second  dilution  of  1  :  400  and  a  third 
of  1  :  800  are  also  prepared. 

As  a  control,  an  equal  quantity  of  the  inoculated  peptone  water 
is  mixed  with  normal  serum  (rabbit  or  horse  serum),  the  dilution 
being  1  :  100. 

In  a  few  minutes  a  distinct  difference  is  observable.  The 
"  control "  shows  with  the  oil-immersion  lens  a  few  isolated  non- 
mobile  bacteria,  while  the  plague-serum  dilution  1  : 200  shows 
larger  and  smaller  masses  of  agglutinated  bacteria. 

After  two  hours'  incubation  the  same  result  is  obtained  with 
the  plague-serum  dilution  of  1  :  400.  No  agglutination,  however, 
is  observed  after  incubation  for  twenty -four  hours  of  the  dilution 
of  1  :  800.  This  agglutination  reaction,  in  conjunction  with  other 
suspicious  phenomena,  justifies  an  official  notification  of  suspected 
plague. 

In  the  examination  of  rats  suspected  to  be  suffering  from  plague 
infection,  it  is  essential  not  only  to  take  the  naked-eye  characters 
into  account,  but  to  make  microscopical  preparations  and  cultures, 
and  to  test  the  cultures  by  animal  inoculations.  Care  must  be  taken 
not  to  mistake  Jicemorrhagic  septiccemic  bacilli  (see  pp.  392,  405)  and 
other  organisms  for  the  plague  bacillus.  The  B.  coli,  B.  proteus,  and 
other  organisms  are  recorded  by  Klein  (loc.  cit.)  as  simulating  the 
B.  pestis. 

Chicken  Cholera 

Chicken  cholera  is  a  disease  of  poultry  characterised  by  profuse 
diarrhoea  ;  its  course  may  be  very  rapid,  and  the  bird  found  dead 
without  having  shown  signs  of  illness.  The  organism  is  a  very 
short  rod,  non-motile,  so  short  that  it  is  almost  ovoid,  0-6  to  0-8  p 
in  length,  and  0-4  to  0-5  p.  in  diameter.  It  stains  by  the  ordinary 
anilin  dyes,  but  not  by  Gram's  method,  and  the  staining  tends  to 
be  polar,  so  that  Pasteur,  who  first  investigated  the  disease, 
described  it  as  a  diplococcus  (Plate  XV.  b).  The  organism  grows 

1  Centralbl.f.  Balct,,  xli  (Originate),  1906,  p.  860, 


CHICKEN  CHOLEKA  405 

freely  on  the  various  culture  media  from  20°  to  38°  C.,  on  agar 
forming  a  thick,  moist,  cream-coloured  layer,  on  gelatin  a  shining, 
white,  expansive  growth  without  liquefaction.  In  broth  a  general 
turbidity  forms,  but  growth  on  potato  is  indifferent.  It  produces 
acid,  does  not  ferment  glucose  or  lactose,  is  aerobic  and  faculta- 
tively anaerobic,  does  not  form  spores,  and  is  killed  by  a  tempera- 
ture of  60°  C.  in  fifteen  minutes.  If  dried  it  dies  in  a  few  days,  but 
retains  its  vitality  for  a  considerable  time  in  damp  earth  or  in  water, 
and  so  infection  is  readily  conveyed.  Fowls  die  after  subcutaneous 
intramuscular  or  intravenous  inoculation  and  by  feeding,  the 
organisms  being  found  abundantly  in  the  blood.  Post-mortem,  the 
serous  membranes  may  be  inflamed  and  haemorrhagic,  the  liver 
large  and  soft,  and  the  intestine  shows  haemorrhagic  spots,  and  is 
sometimes  ulcerated  and  contains  a  mucoid  fluid  stained  with  blood. 
Other  birds,  pigeons,  pheasants,  sparrows,  wild  and  domestic  ducks 
are  also  susceptible  to  the  disease,  and  rabbits  and  guinea-pigs  can 
be  successfully  inoculated  ;  in  the  latter  animal  a  local  abscess 
sometimes  forms  instead  of  a  general  infection.  By  continuous  culti- 
vation with  free  access  of  oxygen  the  virus  becomes  attenuated,  and 
Pasteur  was  able  thus  to  prepare  a  vaccine  which  protected  fowls. 

The  bacillus  of  chicken  cholera  belongs  to  the  group  of  hcemor- 
rhagic  septiccemic  bacilli  (p.  392),  and  seems  to  be  identical  with 
Koch's  bacillus  of  rabbit  septicaemia,  and  with  the  bacillus  of  swine 
plague  (see  p.  373).  These  organisms  tend  to  form  a  stalactite 
growth  in  butter  broth. 

Organisms  have  been  described  by  Klein  in  fowl  enteritis,  grouse 
disease,  etc.,  differing  somewhat  from  the  bacillus  of  chicken 
cholera. 

Mouse  Septicaemia 

This  disease  may  be  conveniently  described  here.  Koch  first 
obtained  a  minute  bacillus  by  injecting  putrefying  material  sub- 
cutaneously  into  mice.  It  seems  to  be  identical  with  the  bacillus 
found  in  swine  erysipelas.  The  organisms  are  met  with  in  large 
numbers  in  the  blood  and  tissues  of  mice.  They  measure  only 
1  p  in  length,  and  occur  in  considerable  numbers  in  the  leucocytes. 
The  bacillus  stains  well  by  Gram's  method,  and  is  stated  by  some 
writers  to  be  motile.  It  grows  readily,  forming  on  agar  extremely 
delicate,  almost  invisible  colonies  ;  in  stab  gelatin  cultures  after 
some  time  a  delicate  cloudiness  radiates  from  the  central  puncture. 
From  an  agar  culture  the  bacilli  are  somewhat  larger  than  those 
found  in  the  animal  body,  and  form  filaments.  It  is  pathogenic  for 
swine,  rabbits,  and  mice. 


CHAPTER  XII 
PNEUMONIA,  INFLUENZA,  AND  WHOOPING-COUGH 

Pneumonia 

PNEUMONIA  is  of  two  types,  lobular,  catarrhal,  or  broncho - 
pneumonia,  and  lobar  or  croupous  pneumonia.  The  former  may 
be  primary,  or  may  be  secondary  and  arise  in  connection  with  many 
of  the  specific  fevers,  as  in  measles,  whooping-cough,  diphtheria, 
enteric  fever,  influenza,  plague,  etc.  The  broncho -pneumonia 
occurring  in  the  course  of  other  diseases  may  be  due  to  the  causative 
organism  of  the  disease,  or  may  be  due  to  other  organisms.  Eyre  1 
examined  62  cases  of  broncho -pneumonia  occurring  in  the  course 
of  other  diseases  and  102  cases  in  which  the  broncho-pneumonia 
was  the  primary  lesion.  Of  these  164  cases,  52-4  per  cent,  yielded 
pure  cultivations  of  some  one  or  other  of  six  bacteria — pneumo- 
coccus,  Strep,  longus,  M.  pyogenes  var.  aureus,  M.  catarrhalis, 
B.  pneumonias,  and  B.  inftuenzce  ;  whilst  47-5  per  cent,  gave  a  mixed 
growth  of  one  or  more  of  these  six  in  association  with  one  or  more 
of  five  other  bacteria — M.  tetragenus,  B.  pertussis,  B.  pyocyaneus, 
B.  typliosus,  B.  diphtherice.  The  B.  coli  also  occurs  in  broncho- 
pneumonia.  Acute  croupous  or  lobar  pneumonia  in  many  of  its 
characters  resembles  an  acute  specific  infection,  and  while  frequently 
a  primary  disease,  may  also  occur  secondarily  in  almost  any  con- 
dition, and  occasionally  in  epidemic  form. 

Friedlander  in  1882-83  first  described  organisms  in  cases  of 
pneumonia. 

In  1883-85  Talamon,  Klein  and  Sternberg  each  described  in 
pneumonic  sputum  an  oval  encapsuled  organism,  which  induced 
pneumonia  in  animals  ;  it  was  termed  by  the  former  the  Micro- 
coccus  lanceolatus,  and  by  Sternberg  the  Micrococcus  Pasteuri. 
This  and  Friedlander's  organisms  were  at  first  believed  to  be 
identical,  but  Frankel  and  Weichselbaum  subsequently  showed  that 

1  Journ.  Path,  and  Bact.,  vol.  xiv,  1910,  p.  160. 
406 


THE  PNEUMOCOOCUS  407 

they  are  quite  distinct,  and  that  the  former  is  the  etiological  agent 
of  acute  croupous  pneumonia. 

The  majority  (95  per  cent.)  of  cases  of  acute  croupous  pneumonia 
are  caused  by  the  Streptococcus  pneumonice,  and  Friedlander's 
organism,  now  termed  Friedlander's  pneumo-bacillus,  or  B.  pneu- 
monice, is  of  etiological  significance  in  only  a  small  minority,  if  at 
all.  The  latter  is,  however,  associated  with  certain  pathological 
processes  which  will  be  referred  to  below. 

From  pleuro-pneumonia  of  cattle,  Nocard  and  Roux  succeeded 
in  cultivating  in  broth  in  collodion  sacs  in  the  peritoneal  cavity  of 
rabbits  an  organism  just  visible  as  minute  granules  with  a  magnifica- 
tion of  2000  diameters.  Bordet  x  states  that  it  may  be  grown  on 
the  medium  employed  by  him  for  the  cultivation  of  the  B.  pertussis 
(p.  417),  and  then  appears  as  fine,  straight,  curved,  undulating,  or 
even  spirillar  filaments  not  unlike  spirochaetes. 


The  Streptococcus  (Diplococcus)  pneumoniae 

Synonyms,  Frankel's  pneumococcus,  Micrococcus  Pasteuri  (Stern- 
berg),  Micrococcus  lanceolatus  (Talamon),  Micrococcus  pyogenes 
tenuis  (Rosenbach). 

Morphology. — The  Streptococcus  pneumonice  in  the  sputum 
and  tissues  occurs  as  an  oval  or  lance- shaped  coccus  united 
in  pairs,  occasionally  in  chains  of  three  or  four  elements, 
and  then  often  almost  spherical,  and  is  generally  surrounded 
by  a  well-marked  capsule  (Plate  XVI.  a).  In  order  to 
isolate  the  organism  several  tubes  of  glycerin  agar,  serum 
or  serum- agar  may  be  inoculated  with  rusty  sputum  and 
incubated  for  forty- eight  hours ;  in  some  a  pure  culture 
may  be  obtained.  A  more  certain  method  is  to  inject 
a  drop  or  two  of  the  rusty  sputum  into  the  peritoneal 
cavity  of  a  mouse  or  young  rabbit.  The  animal  will  die 
in  from  twenty-four  to  thirty-six  hours,  and  the  organism 
will  be  found  in  considerable  numbers  in  the  lung  and 
blood,  from  which  cultures  may  be  obtained.  It  is  non- 
motile,  stains  with  the  ordinary  anilin  dyes  and  by  Gram's 
method. 

1  Ann.  de  VInst.  Pasteur,  xxiv,  1910.  March. 


408  A  MANUAL  OF  BACTERIOLOGY 

Cultural  characters.- — The  S.  pneumonice  is  aerobic  and 
almost  facultatively  anaerobic.  On  glycerin  agar  at  37°  C. 
it  forms  minute,  transparent,  almost  invisible  colonies  like 
droplets  of  fluid  ;  on  serum  the  growth  has  much  the  same 
characters,  but  is  somewhat  more  abundant.  It  hardly 
grows  on  gelatin  at  the  ordinary  temperature,  but  in  a 
20  per  cent,  gelatin  at  25°  C.  minute  white  colonies  develop 
without  liquefaction.  In  broth  it  produces  a'  slight  cloudi- 
ness ;  it  does  not  grow  on  potato  but  develops  in  milk, 
which  is  usually  coagulated ;  neutral  litmus  glucose- agar 
becomes  red  during  growth,  indicating  the  production  of 
acid.  The  fermentation  reactions  are  given  in  the  Table 
on  p.  235.  Hiss's  medium  (p.  291)  with  inulin  is  fermented 
and  coagulated  ;  most  other  streptococci  fail  to  ferment 
inulin.  On  the  ordinary  culture  media  it  retains  its 
vitality  for  a  short  time  only,  not  more  than  about  a  week  ; 
but  if  a  little  blood  be  smeared  over  the  surface  of  the 
agar  the  vitality  may  be  prolonged  for  a  month  or  even 
longer.  Washbourn  recommended  an  agar  rendered  alka- 
line to  the  extent  of  4  c.c.  of  normal  caustic  soda  per  litre, 
after  neutralisation,  rosolic  acid  being  the  indicator.  This 
medium  is  smeared  with  blood,  placed  in  the  incubator 
for  twenty-four  hours  to  ascertain  whether  it  be  sterile, 
then  inoculated,  capped,  and  kept  at  37°  C.  Foa's  method 
for  keeping  Frankel's  pneumococcus  alive  and  virulent  is 
to  receive  the  infected  blood  of  an  inoculated  animal  into 
a  small  glass  tube  5  mm.  in  diameter  and  20  cm.  long,  so 
that  the  blood  completely  fills  the  tube,  which  is  then 
sealed  and  kept  away  from  the  light  at  the  ordinary  tem- 
perature. If  inoculated  on  to  ordinary  gelatin,  which  is 
then  kept  in  the  blood  heat  (37°  C.)  incubator,  the  organism 
retains  its  vitality  for  a  month  or  six  weeks. 

Under  cultivation  the  S.  pneumonice  usually  assumes 
the  form  of  a  short  streptococcus  (Plate  XVI.  b)  (included 
by  Gordon  in  his  S.  brevis  class)  and  the  capsule  is  lost. 


PLATE  XVI. 


Diplococcus  pneumonia.     Film  preparation  of  blood  of 
inoculated  animal.      X  1000. 


b.  Diplococcus  pnzumonice.     Film  preparation  of  a  pure 
culture.      X  1500. 


THE  PNEUMOCOCCUS  409 

but  is  regained  on  passage  through  a  susceptible  animal, 
or  by  growing  in  fluid  serum.  A  good  deal  of  variation 
occurs  in  the  morphology  of  the  organism  obtained  from 
different  sources  and  under  cultivation.  The  thermal 
death-point  of  the  S.  pneumonia  according  to  Sternberg 
is  52°  C.,  the  time  of  exposure  being  ten  minutes,  and  it  is 
readily  destroyed  by  the  ordinary  germicides,  by  light,  and 
by  desiccation ;  but  in  dried  sputum  it  may  retain  its 
vitality  and  virulence  unimpaired  for  weeks. 

Pathogenic  action. — The  S.  pneumonia  is  pathogenic  for 
a  number  of  animals,  the  most  susceptible  being  mice, 
then  in  decreasing  order,  rabbits,  rats,  guinea-pigs,  and 
dogs.  Pigeons  and  fowls  are  immune.  Death  follows 
after  subcutaneous,  intravenous,  intraperitoneal,  or  intra- 
thoracic  injection  of  a  virulent  culture,  or  of  rusty  pneu- 
monic sputum,  into  mice  and  rabbits  in  twenty-four  to 
forty-eight  hours.  The  virulence  of  the  organism  varies 
considerably ;  under  cultivation  it  may  be  completely 
lost,  while  by  a  series  of  passages  through  a  susceptible 
animal  it  may  be  much  increased.  The  less  virulent  it  is 
the  longer  it  tends  to  retain  its  vitality  under  cultivation. 
Except  when  injected  into  the  lung  or  into  the  trachea, 
pneumonia  does  not  result,  but  the  disease  runs  the  course 
of  a  septicaemia  with  high  temperature  and  dyspnoea, 
death  being  generally  preceded  by  a  subnormal  temperature 
and  often  convulsions.  The  post-mortem  appearances  are 
much  oedema  and  inflammatory  infiltration  at  the  seat  of 
inoculation,  hemorrhages  in  the  serous  membranes,  enlarge- 
ment and  congestion  of  the  spleen,  and  congestion  of  the 
lungs.  The  organisms  occur  in  large  numbers  in  the 
blood,  lungs,  and  spleen,  usually  in  the  form  of  oval 
diplococci  with  well-marked  capsules  (Plate  XVI.  a),  but 
sometimes  as  short  chains  of  streptococci.  When  injected 
into  the  lung  or  trachea  a  typical  fibrinous  or  croupous 
pneumonia  results. 


410  A  MANUAL  OF  BACTERIOLOGY 

The  S.  pneumonia  is  the  cause  of  acute  croupous  pneu- 
monia in  man,  and  occurs  in  large  numbers  in  the  rusty 
sputum  and  hepatised  lung,  and  in  20  per  cent,  of  the 
cases  can  be  isolated  from  the  blood  if  5-10  c.c.  be  cultured. 
The  production  of  a  typical  pneumonic  process  experi- 
mentally and  the  presence  of  the  diplococcus  in  a  large 
proportion  of  cases  of  acute  croupous  pneumonia  point 
to  its  specific  relationship  to  the  disease.  With  regard 
to  the  latter  observation,  Weichselbaum  obtained  it  in 
94  cases  out  of  129  examined,  Wolf  in  66  out  of  70  cases, 
and  Netter  in  75  per  cent,  of  the  cases  examined.  In 
America  the  disease  has  of  late  been  much  on  the  increase, 
in  Chicago  the  mortality  having  reached  as  high  as  20 
per  10,000  inhabitants.  Acute  croupous  pneumonia  some- 
times occurs  in  epidemic  form  and  has  decimated  the 
native  labourers  in  the  Rand  mines. 

The  organism  is  frequently  present  in  the  saliva  of 
healthy  individuals,  as  shown  by  Netter,  Sternberg,  and 
others,  and  the  generally  accepted  idea  of  the  relationship 
of  "  catching  cold  "  to  an  attack  of  the  disease  is  explicable 
on  the  theory  that  the  action  of  cold  lowers  vitality,  and 
renders  the  tissues  vulnerable  to  the  attacks  of  the  organism 
already  in  close  proximity  to  them. 

Besides  acute  croupous  pneumonia,  more  than  half 
the  cases  of  broncho-pneumonia,  both  primary  and 
secondary  in  the  course  of  other  diseases,  are  due  to  the 
S.  pneumonia,  which  is  also  associated  with  a  number  of 
other  important  pathological  conditions  in  man.  It  is  a 
pyogenic  organism,  producing  abscesses  when  inoculated 
into  a  relatively  insusceptible  animal  such  as  a  dog,  and 
has  been  met  with  in  abscesses,  empyema,  suppuration 
in  the  antrum,  and  purulent  arthritis.  It  is  also  found 
in  about  half  the  cases  of  purulent  meningitis,  sometimes 
in  cerebro-spinal  meningitis,  in  about  a  third  of  the  cases 
of  otitis  media  and  infective  endocarditis,  sometimes  in 


ANTI-PNEUMOCOCCIC  SERUM  411 

purulent  pericarditis,  and  occasionally  in  peritonitis.  The 
pneumococcus  is  also  frequent  in  chronic  bronchial  catarrh. 
An  agglutination  reaction  with  patient's  serum  on  the 
pneumococcus  is  only  very  irregularly  obtained  and  normal 
serum  rarely  exerts  any  bactericidal  effect  upon  the 
organism. 

As  regards  opsonic  determinations,  freshly  isolated 
strains  frequently  fail  to  give  any  phagocytosis,  and  every 
strain  of  pneumococcus  gives  a  different  amount  of  phago- 
cytosis. For  the  control,  the  pooled  serum  of  several 
individuals  should  be  used,  and  the  culture  should  be 
emulsified  in  distilled  water.  The  serum  of  the  Rand 
native  seems  to  have  a  very  low  opsonic  content  for  the 
pneumococcus  compared  with  that  of  the  European.1 

Toxins. — Auld  separated  a  proteose  and  an  organic  acid 
from  the  blood  and  organs  of  infected  animals,  and  from 
cultivations  of  the  S.  pneumonia  in  alkali- albumin  the 
same  products  were  apparently  obtained,  the  alkaline 
medium  soon  becoming  permanently  acid.  The  proteose 
on  subcutaneous  or  intravenous  injection  produced  some 
fever  ;  on  intra- thoracic  injection  fever  and  dyspnoea,  and 
post-mortem  pleurisy  and  consolidation  of  the  lung  were 
found.  The  organic  acid  produced  slight  rise  of  tempera- 
ture, but  no  other  symptom.  Macfadyen 2  obtained  an 
endotoxin  by  triturating  cultures  with  liquid  air. 

Anti-serum. — Immunity  can  be  conferred  on  susceptible 
animals  by  treating  them  with  attenuated  cultures,  or 
by  inoculation  with  increasing  doses  of  filtered  broth 
cultures  of  the  virulent  organism  followed  by  doses  of  the 
living  organism.  The  blood-serum  of  such  immunised 
animals  will  protect  other  animals  when  injected,  and  an 
anti-pneumococcic  serum  has  been  prepared  by  the  fore- 
going method.  This  anti-serum  has  been  used  in  the 

1  Wright,  Lancet,  1914,  i,  p.  1  et  seq. 

2  Brit.  Med.  Journ.,  1906,  vol.  ii,  p.  776  (Refs.). 


412  A  MANUAL  OF  BACTERIOLOGY 

treatment  of  pneumonia  and  other  pneumococcic  infec- 
tions, but  the  results  have  not  been  very  encouraging. 
The  protective  serum  seems  to  produce  aggregation  of 
the  cocci  when  added  to  a  culture  of  the  diplococcus. 
Klemperer  and  Washbourn  found  that  the  serum  of  con- 
valescent patients  possesses  some  degree  of  protective 
power.  The  serum,  however,  withdrawn  during  the 
pyrexial  stage  of  the  disease  rather  increases  the  suscepti- 
bility of  animals  to  pneumococcic  infection. 

Vaccine. — A  vaccine  prepared  from  cultures  killed  by 
heat  and  standardised  has  been  found  of  service  in  chronic 
pneumococcic  infections,  and  has  also  been  employed  in 
acute  croupous  pneumonia.1  Wright  (loc.  cit.)  has  also 
recommended  a  vaccine  for  prophylactic  inoculation  against 
pneumonia  on  the  Rand,  a  dose  of  1000  millions  apparently 
being  the  optimum  for  this  purpose. 

Friedlander's  Pneumo-bacillus 

This  organism,  already  referred  to  above  in  the  general 
discussion  of  pneumonia,  and  originally  believed  by  Fried- 
lander  to  be  the  cause  of  the  disease,  has  been  obtained 
by  recent  observers  in  only  a  small  proportion  of  cases 
of  pneumonia. 

Morphology. — The  B.  pneumonia  is  a  very  pleomorphic 
organism,  occurring  in  sputum  or  in  the  blood  of  an  inocu- 
lated animal  generally  as  a  short  rod  with  rounded  ends 
surrounded  by  a  marked  capsule.  It  is  non-motile,  does 
not  form  spores,  and  is  readily  stained  with  the  ordinary 
anilin  dyes,  but  not  by  Gram's  method — an  important 
distinction  from  the  S.  pneumonia.  In  cultivations  it 
forms  short  rods,  long  rods,  chains,  and  even  filaments, 
the  capsule  being  absent,  but  this  is  regained  on  passage 
through  a  susceptible  animal. 

1  Willcox  and  Morgan,  Brit.  Med.  Journ.,  1909,  vol.  ii,  p.  1050. 


THE  PNEUMO-BACILLUS 


413 


Cultural  characters. — The  B.  pneumonice  is  aerobic  and 
facultatively  anaerobic,  and  may  produce  indole.  It  grows 
readily  on  the  various  culture  media  from  20°  to  37°  C., 
on  agar  and  blood- serum  forming  a  copious,  viscid,  greyish 
growth ;  on  gelatin,  a  thick,  white,  shining,  porcelain- like 
growth  without  liquefaction ; 
and  in  stab- cultures  in  gelatin 
a  so-called  nail-shaped  growth 
is  developed  (Fig.  46),  consist- 
ing of  a  white  growth  along 
the  needle- track,  tapering  from 
above  downwards,  and  at  the 
surface  heaped  up  and  ex- 
panded, forming  the  "  head  "  of 
the  nail.  On  potato  a  copious 
whitish  growth  develops,  while 
milk  is  curdled  and  gas- bubbles 
frequently  form  in  stab- gelatin 
cultures.  It  is  an  active  fer- 
menter  of  carbohydrates ;  the 
fermentation  reactions  are  given 
in  the  Table,  p.  381. 

Pathogenic    action. — The 

pneumo- bacillus  of  Friedlander     FIG.  46. — Friedlander's  pneumo- 

is     pathogenic    to    mice     and       bacillus'     Gelatin    stab-cul- 

^        .  &  .  ture,  seven  days  old. 

guinea-pigs,    but    rabbits    are 

immune.  Post-mortem,  the  spleen  is  enlarged,  the  lungs 
are  congested  and  consolidated  in  patches,  and  the  organism 
is  found  in  large  numbers  in  the  blood.  In  a  small  per- 
centage of  cases  of  croupous  pneumonia  Friedlander's 
bacillus  may  be  associated  with  the  S.  pneumonice.  Fried- 
lander's  bacillus  may  sometimes  set  up  a  broncho-pneu- 
monic or  bronchitic  process,  and  is  occasionally  associated 
with  anginal  conditions,  which  are  characterised  by  the 
formation  of  a  false  membrane,  with  an  absence  of  any 


414  A  MANUAL  OF  BACTERIOLOGY 

general  symptoms.  A  microscopical  examination  of  the 
membrane  will  show  the  organisms  surrounded  with  a 
capsule  and  unstainable  by  Gram's  method.  If  a  culture 
be  made  on  serum,  the  large,  round,  greyish  colonies  of 
the  bacillus  will  be  recognisable  in  fifteen  to  twenty  hours, 
and  should  be  examined  microscopically.  To  obtain  a 
pure  culture  a  white  mouse  should  be  inoculated  from  a 
colony  ;  it  will  die  in  twenty- eight  to  sixty  hours.  Fried- 
lander's  pneumo- bacillus  has  also  been  met  with  in  water 
by  Grimbert.  According  to  him,  it  is  identical  with  the 
B.  capsulatus  of  Mori. 


Clinical  Examination  (Pneumonia) 

1.  Make   smear  specimens  from   the  rusty  sputum,  and  stain 
some  with  Loffler's  blue,  and  others  by  Gram's  method  with  eosin. 
By  a  microscopical  examination  the  oval  diplococci  will  be  readily 
recognised,  the  B.  pneumonia  and  B.  pestis  being  distinguished 
from  the  S.  pneumonia  by  being  decolorised  by  Gram's  method. 
The  latter  organism  is  the  only  one,  moreover,  which  is  likely  to 
be  ordinarily  met  with. 

2.  If  the  diplococci  are  found  to  be  fairly  abundant  in  the  sputum, 
and  other  organisms  nearly  absent,  an  attempt  may  be  made  to 
cultivate   by  inoculating  several  glycerin-agar  and  serum  tubes 
and  incubating  at  37°  C.  for  forty-eight  hours. 

3.  If  the  diplococci  are  scanty,  or  so  mixed  with  other  organisms 
that  it  is  difficult  to  identify  them,  and  probably  impossible  to 
obtain  a  pure  culture,  a  drop  or  two  of  the  sputum  should  be  injected 
into  the  peritoneal  cavity  of  a  mouse  or  rabbit.     The  animal  will 
die  in  from  twenty -four  to  thirty-six  hours,  and  the  S.  pneumonice 
will  be  found   plentifully  in  smears  prepared  from  the  blood  or 
lung-juice,  and  pure  cultures  can  be  readily  obtained  by  inoculating 
glycerin-agar  tubes  with  the  blood  or  lung-juice. 

4.  The    culture   or   inoculation   method,    preferably   both,    will 
probably  have  to  be  adopted  for  the  recognition  and  isolation  of 
the  S.  pneumonice  in  pus  from  empyemata,  abscesses,  etc. 

5.  Friedlander's    pneumo-bacillus    can    be    readily   isolated    by 
making  gelatin-plate  cultivations,  in  which  its  colonies  form  white, 
shining,  heaped-up  points. 


INFLUENZA  415 

Influenza 

A  minute  bacillus  was  first  described  in  this  disease 
by  Pfeiffer  in  1892,  who  found  it  in  large  numbers  in  the 
bronchial  secretion.  In  order  to  isolate  the  organism  a 
patient  with  bronchial  expectoration  should  be  chosen ; 
he  rinses  his  mouth  and  gargles  his  throat  with  hot  water 
several  times,  and  then,  after  coughing,  the  expectoration 
is  obtained.  A  little  of  this  expectoration  is  washed  by 
shaking  in  a  test-tube  with  sterile  salt  solution,  then 
repeating  the  washing  with  sterile  salt  solution  in  a  second 
and  finally  in  a  third  test-tube.  By  means  of  a  platinum 
needle  a  number  of  glycerin- agar  and  blood- agar  tubes 
are  inoculated  with  the  sputum  after  the  last  washing,  and 
incubated  at  37°  C. 

Morphology. — The  influenza  bacillus  is  one  of  the  smallest 
bacilli  with  which  we  are  acquainted.  It  is  a  minute  rod 
0-5-1-5  jji  in  length,  and  is  non-motile  and  non-sporing. 
It  does  not  stain  by  Gram's  method,  and  not  very  readily 
with  the  ordinary  dyes,  dilute  carbol-fuchsin  or  prolonged 
staining  with  Loffler's  blue  yielding  the  best  results,  the 
poles  tending  to  stain  more  deeply  than  the  centre.  In 
the  sputum  it  occurs  singly,  in  short  chains,  in  small  groups, 
or  in  larger  masses,  being  most  numerous  early  in  the 
acute  stage  of  the  disease. 

Cultural  characters. — The  bacillus  is  strictly  aerobic, 
and  no  growth  occurs  on  media  at  22°  C.  On  glycerin- 
agar  and  blood-serum  at  37°  C.  it  forms  very  small,  trans- 
parent, drop-like  colonies  in  from  twenty-four  to  forty- 
eight  hours,  which,  according  to  Kitasato,  never  became 
confluent.  There  is  no  growth  on  potato.  The  organism 
grows  best  on  media  containing  blood,  such  as  agar  smeared 
with  sterile  human,  rabbit's,  or  pigeon's  blood.  In  broth 
it  grows  at  the  surface  in  fine  white  flakes  which  subse- 
quently sink. 


416  A  MANUAL  OF  BACTERIOLOGY 

It  soon  dies  out  in  cultivation,  but  according  to  Klein 
can  be  kept  alive  for  some  weeks  in  gelatin  incubated  at 
37°  C.  The  melted  gelatin  remains  clear,  the  growth 
forming  a  delicate  flocculent  precipitate  at  the  bottom. 
Preparations  from  cultures  show  long  twisted  chains  and 
threads  of  bacilli,  aggregated  so  as  to  form  dense  networks 
and  convolutions.  These  chains  or  threads  are  composed 
of  bacilli  placed  end  to  end,  and  united  by  a  continuation 
of  the  cell- membrane.  Involution  forms  occur.  It  is 
stated  to  grow  better  in  association  with  the  M.  pyogenes 
var.  aureus  than  alone.  The  organism  does  not  seem  to 
be  able  to  live  outside  the  body  for  any  length  of  time, 
and  is  readily  destroyed  by  desiccation,  weak  antiseptics, 
and  by  a  temperature  of  60°  C.  acting  for  five  minutes. 

Pathogenic  action. — Canon  stated  that  he  obtained  this 
bacillus  from  the  blood  in  a  number  of  cases,  but  many 
other  investigators  have  failed  to  find  it.  Klein  also 
obtained  it  in  six  cases  out  of  forty-three  examined. 
According  to  Pfeiffer  the  bacillus  is  pathogenic  only  to 
monkeys  and  rabbits.  Klein,  however,  was  unable  to 
obtain  any  definite  effects  in  these  animals  by  the  injection 
either  of  sputum  rich  in  bacilli  or  of  pure  cultures. 

The  influenza  bacillus  is  met  with  in  all  uncomplicated 
cases  of  influenza  in  the  nasal  and  bronchial  secretions, 
often  almost  in  pure  culture,  and  in  the  bronchial  tubes 
and  lung  in  the  pneumonic  complications  accompanying 
the  disease.  The  organisms  disappear  with  convalescence, 
and  are  not  met  with  in  other  diseases.  Klein  x  appears 
to  consider  that  the  pneumonia  often  complicating  the 
disease  is  probably  directly  due  to  the  bacillus.  The 
typical  influenza  pneumonia  is  of  the  lobular  type  with  a 
cellular  rather  than  a  fibrinous  exudate.  True  lobar 
pneumonia,  due  to  the  S.  pneumonia,  may,  however, 

1  "  Further  Report  on  Epidemic^ Influenza,"  1889-92,  Loc.  Gov. 
Board  Report,  1893,  p.  85. 


PERTUSSIS  417 

often  complicate  the  influenzal  attack.  The  organism  also 
occurs  in  bronchitis,  broncho-pneumonia,  and  whooping- 
cough. 

Although  the  typical  influenza  may  be  due  to  the  B.  influenzce, 
many  febrile  conditions  attended  with  pulmonary  catarrh  and 
frequently  termed  "  influenza  "  are  not  due  to  this  organism.  In 
an  epidemic  simulating  influenza  occurring  in  Essex  in  1905,  the 
examination  was  negative  as  regards  streptococci,  B.  diphtherice 
and  B.  influenzce,  but  the  M.  catarrhalis  was  present  in  number  in 
most  cases  (twenty-two  out  of  twenty-four).  This  organism  was 
originally  isolated  by  Seifert  in  a  small  epidemic  of  infectious 
bronchitis,  afterwards  by  Pfeiffer  in  cases  of  broncho-pneumonia  in 
young  children  (see  p.  248).  Two  other  Gram-negative  cocci  were 
also  isolated  from  three  other  cases  (see  Table,  p.  248). 


Clinical  Examination 

In  cases  of  influenza,  accompanied  with  bronchitis  or  pneumonia, 
the  influenza  bacillus  may  be  met  with  in  large  numbers  in  the 
sputum,  and  their  presence  may  aid  in  confirming  the  diagnosis. 
Film  preparations  may  be  stained  with  carbol-methylene  blue. 


Whooping-cough  (Pertussis)  l 

An  influenza-like  bacillus  has  been  isolated  by  Koplik,  Czaplewski 
and  Hensel,  Davis  and  others  in  this  disease,  but  the  researches  of 
Bordet  and  Gengou  have  shown  that  it  is  distinct  from  the  influenza 
bacillus. 

The  B.  pertussis  is  a  minute  baciUus,  very  like  the  B.  influenzce, 
non-motile,  non-sporing,  and  Gram-negative.  It  is  scanty  in  the 
bulk  of  the  expectoration,  but  is  abundant  in  the  viscid  exudate, 
rich  in  leucocytes,  coming  from  the  depth  of  the  bronchi,  and  voided 
at  the  end  of  a  paroxysm  of  coughing. 

It  is  best  isolated  on  a  medium  consisting  of  defibrinated  blood 
(human  or  rabbit),  thoroughly  mixed  with  an  equal  volume  of  3  per 
cent,  agar  containing  a  little  extract  of  potato  made  with  4  per 
cent,  aqueous  glycerin.  It  forms  on  this  a  fairly  thick  whitish 
streak,  the  subjacent  blood  being  hsemolysed.  It  may  also  be 

1  See  Bordet,  Brit.  Med.  Journ.,  1909,  vol.  ii,  p.  1062. 

2? 


418  A  MANUAL  OF  BACTERIOLOGY 

grown  in  serum  or  blood  broth  in  shallow  layers.  After  acclimatisa- 
tion to  artificial  media  it  will  develop  on  the  ordinary  laboratory 
media. 

The  B.  pertussis  is  agglutinated  feebly  by  the  blood  of  patients, 
but  complement-fixation  is  marked. 

Monkeys  are  stated  to  develop  a  typical  whooping-cough  on 
inoculation,  but  the  ordinary  laboratory  animals  are  susceptible 
only  to  massive  intraperitoneal  or  intravenous  inoculation,  death 
ensuing  from  a  septicaemic  process. 

Attempts  have  been  made  to  treat  the  disease  with  a  vaccine. 


CHAPTER  XIII 
ANAEROBIC  ORGANISMS 

TETANUS— MALIGNANT  (EDEMA— BLACK  QUARTER- 
BACILLUS  WELCHII  (AEROGENES  CAPSULATUS,  EN- 
TERITIDIS  SPOROGENES)  —  BACILLUS  CADAVERIS 
SPOROGENES— CLOSTRIDIUM  BUTYRICUM 

Tetanus 

THE  causation  of  tetanus  was  for  a  long  time  involved  in  mystery. 
No  obvious  or  characteristic  changes  being  met  with  after  death, 
the  disease  was  regarded  by  many  as  "functional."  Others 
believed  that  a  primary  lesion  of  the  central  nervous  system  might 
be  the  cause  of  the  affection,  while  a  few  classed  it  with  the  specific 
diseases. 

It  had  long  been  noticed  that  wounds  soiled  with  earth  were 
specially  prone  to  be  followed  by  tetanus,  and  Sternberg  in  1880, 
and  Nicolaier  in  1884,  produced  tetanus  in  rabbits  by  introducing 
a  little  garden  earth  beneath  the  skin.  The  latter  observer  found 
at  the  seat  of  inoculation  and  in  his  impure  cultures — for  he  was 
unable  to  obtain  pure  ones — a  distinctive  bacillus,  and  he  was  able 
with  these  cultures,  and  with  the  pus  from  the  seat  of  inoculation, 
to  induce  tetanus  in  other  animals.  Carle  and  Rattone  subsequently 
showed  that  the  bacillus  of  Nicolaier  was  present  in  the  tissues  of, 
and  secretions  from,  the  wound,  in  cases  of  traumatic  tetanus  in 
man,  and  that  inoculation  with  the  pus  from  such  a  wound  pro- 
duced tetanus  in  the  lower  animals — observations  which  were  con- 
firmed by  Rosenbach  in  1885.  The  bacillus  was  isolated  in  pure 
culture  by  Kitasato  in  1889  by  taking  the  impure  cultures  obtained 
from  the  wound  in  a  case  of  traumatic  tetanus,  heating  to  80°  C., 
and  plating  the  heated  cultures,  the  plates  being  incubated 
anaerobically  in  hydrogen. 

419 


420  A  MANUAL  OF  BACTERIOLOGY 


The  Bacillus  tetani 

Morphology. — The  Bacillus  tetani  is  a  straight,  slender 
rod  with  rounded  ends,  but  under  cultivation  the  rods 
may  grow  into  longish  filaments.  It  is  somewhat  motile 
and  possesses  a  large  number  of  flagella,  three  or  four  of 
which  are  generally  thicker  than  the  rest.1  Spores  are 
freely  formed ;  they  are  spherical  and  develop  at  one 
extremity  of  the  rod,  and  their  diameter  being  much 
greater  than  that  of  the  rod,  the  spore-bearing  organism 
has  been  likened  to  a  "  pin "  or  "  drum-stick  "  (Plate 
XVII.  a).  It  stains  with  the  ordinary  anilin  dyes,  and 
also  by  Gram's  method.  "  Drum-stick  "  bacilli  are  not 
necessarily  tetanus  ;  other  anaerobic  bacilli,  e.g.  B.  putri- 
ficus  (coli),  may  also  have  large  terminal  spores. 

Cultural  characters.- — The  B.  tetani  is  a  strictly  anaerobic 
organism,  and  will  not  grow  in  the  presence  of  a  trace  of 
free  oxygen,  nor  in  an  atmosphere  of  carbon  dioxide.  It 
can  be  cultivated  in  deep  stabs  in  glucose  agar  and  gelatin, 
or  in  broth  by  Buchner's  method,  or  in  an  atmosphere  of 
hydrogen  (p.  73).  In  a  gelatin  stab-culture  at  22°  C.  the 
growth  radiates  from  the  central  puncture,  and  the  gelatin 
is  slowly  liquefied.  In  a  glucose  agar  stab- culture  it  forms 
feathery,  radiating  outgrowths  from  the  central  puncture, 
a  small  amount  of  gas  being  formed  (Fig.  47).  Broth 
becomes  turbid  with  the  formation  of  some  gas  and  the 
development  of  a  foul  odour ;  there  is  no  film  formation. 
The  colonies  have  a  central  opaque  portion  surrounded  by 
diverging  rays.  It  grows  on  serum  without  liquefaction 
and  in  milk  without  curdling.  The  tetanus  bacillus  remains 
alive  for  some  time,  possibly  indefinitely,  in  cultures,  and 
the  spores  retain  their  vitality  for  years  in  the  dried  state, 
withstand  a  temperature  of  80°  C.  for  an  hour,  but  are 

1  Kanthack  and  Connell,  Journ.  Path,  and  Bact.,  vol.  iv,  1897,  p.  452. 


THE  TETANUS  BACILLUS 


421 


killed  by  boiling  for  five  minutes.     Carbolic  acid  (1  :  20) 
does  not  destroy  the  spores  under  about  fifteen  hours. 

Occurrence  and  pathogenic  action. — Man  and  the  horse 
are  most  subject  to  tetanus  ;  cattle  and  sheep  are  rarely 
affected,  while  the  fowl,  frog,  triton,  snake  and  tortoise 
are  immune.  Mice,  guinea-pigs  and 
rabbits  are  all  very  susceptible.  The 
bacillus  is  present  in  the  superficial 
layers  of  the  soils  in  many  localities,  but 
not  in  all,  and  this  accounts  for  the  fact 
that  tetanus  is  rare  in  some  places  and 
frequent  in  others.  The  natives  of  the 
Solomon  Islands  have  made  use  of  this 
fact  for  the  preparation  of  poisoned 
arrows.  The  arrows  are  tipped  with  a 
viscid  fluid,  then  rubbed  in  the  soil  from 
a  mangrove  swamp  containing  tetanus 
spores,  and  afterwards  dried.  Individuals 
wounded  with  these  arrows  generally  de- 
velop tetanus. 

Tetanus  spores  are  frequently  present 
in    the    dejecta    of    cattle,    horses,    and 

other   animals,  and   occasionally  of   man 
,        .90v  FIG.  47.— Tetanus 

bacillus.       Stab- 

The  bacillus  is  confined  to  the  seat  culture  in  glucose 
of  inoculation,  or  at  most  is  met  with  afar'  seven  days 
in  the  nearest  lymphatic  glands,  so 
that  the  general  symptoms  are  due  to  the  absorption  of 
toxin.  The  researches  of  Ransom  and  Meyer  have  shown 
that  the  tetanus  toxin  is  mainly  absorbed  by  the  nerve- 
trunks  (see  also  p.  159).  The  organisms  associated  with 
the  tetanus  bacillus  in  earth  are  probably  of  considerable 
importance  in  the  production  of  the  disease,  for  it  has  been 
shown  that  if  the  tetanus  bacilli  and  their  spores  be  care- 
fully washed  so  as  to  remove  all  adherent  toxins,  they  fail 


422  A  MANUAL  OF  BACTERIOLOGY 

to  set  up  tetanus  on  inoculation,  while  if  the  same  washed 
bacilli  be  injected,  together  with  a  little  lactic  acid,  tetanus 
follows,  the  explanation  being  that  the  bacilli  are  unable 
to  multiply  unless  the  surrounding  tissues  are  damaged 
and  phagocytosis  is  prevented.  The  associated  organisms 
in  the  wound  probably  effect  this,  and  do  not  act  by 
producing  a  condition  of  anaerobiosis  as  has  been  suggested. 
Semple  x  has  recently  found  that  tetanus  spores  are  occa- 
sionally present  in  the  human  intestinal  tract  (Hamilton 
suggested  that  tetanoid  organisms  in  the  intestinal  tract 
might  be  the  cause  of  the  so-called  idiopathic  or  rheumatic 
tetanus).  He  injected  guinea-pigs  with  washed  spores, 
and  tetanus  did  not  ensue,  but  the  tissue  at  the  site  of 
inoculation,  examined  five  to  seven  months  later,  still 
contained  the  living  spores.  Semple  suggests  that  such 
latent  spores  may  in  some  instances  be  disturbed  and 
become  active  by  the  hypodermic  or  intra- muscular  injec- 
tion of  quinine,  owing  to  the  tissue  necrosis  and  inhibition 
of  phagocytosis  produced  by  the  drug. 

Toxins.- — Cultivated  anaerobically  in  broth,  the  tetanus 
bacillus  forms  a  most  potent  extra- cellular  toxin,  so  that 
if  the  culture  be  filtered  through  a  porcelain  filter,  0-001  c.c., 
0-0001  c.c.,  or  even  0-00001  c.c.  of  the  filtrate  is  a  fatal  dose 
for  a  guinea-pig. 

Tetanus  toxin  broth  contains  a  tetanising  substance, 
termed  tetano-spasmin,  and  also  a  haemolysin,  tetano- 
lysin.  The  toxin  has  a  special  affinity  for  nerve-tissue 
(see  p.  159).  Injected  into  animals  such  as  the  mouse, 
guinea-pig  and  rabbit,  the  toxin  broth  produces  tonic, 
not  clonic,  spasm  and  with  small  doses  the  muscles  at  or 
near  the  seat  of  inoculation  tend  first  to  be  affected,  so 
that  the  spine  may  be  curved,  the  leg  paralysed,  etc. 
(Fig.  48). 

By  treatment  with  carbon  disulphide,  tetanus  toxin 
1  Sc.  Mem.  Gov.  of  India,  No.  43,  1911. 


TETANUS 


423 


broth  becomes  practically  non-toxic,  though  it  still  retains 
its  power  of  immunising  on  inoculation  and  of  combining 
with  antitoxin — that  is  to  say,  bodies  are  formed  analogous 
to  the  toxoids  of  diphtheria  toxin. 

Brieger,  from  impure  cultures  of  the  tetanus  bacillus, 
obtained  two  basic  bodies  which  he  termed  "  tetanine  " 
a,nd  "  tetano- toxin,"  the  former  producing  tetanic  symp- 


FIG.  48. — Guinea-pig  inoculated  with  a  small  dose  of   tetanus  toxin, 
showing  paralytic  condition  of  right  hind  leg  due  to  spasm. 


toms  in  mice,  and  the  latter  tremor,  paralysis,  and  finally 
convulsions.  Brieger  also  isolated  tetanine  from  the 
amputated  limb  of  a  tetanic  patient.  Brieger  and  Frankel 
obtained  a  tox- albumin  from  bouillon  cultures  which 
induced  tetanus  in  guinea-pigs.  Brieger  and  Cohn  subse- 
quently investigated  the  tetanus  poison  obtained  by  preci- 
pitating veal-broth  cultures  with  ammonium  sulphate 
added  to  saturation,  and  purifying  by  re- dissolving,  preci- 
pitating the  protein  with  basic  lead  acetate,  and  removing 
other  soluble  impurities  by  dialysis.  The  purified  product 
forms  yellow  flakes,  soluble  in  water,  but  not  giving  the 


424  A  MANUAL  OF  BACTERIOLOGY 

Millon  and  xanthoproteic  reactions.  It  is  not  precipitated 
by  most  metallic  salts,  and  is  not  carried  down  by  Roux 
and  Yersin's  method  of  precipitation  with  calcium  phos- 
phate. It  contains  no  phosphorus  and  only  traces  of 
sulphur.  Of  the  most  active  preparation  0-00000005  grm. 
killed  a  mouse. 

In  a  case  of  tetanus  examined  by  Sidney  Martin,  an 
albumose,  chiefly  deutero-albumose,  was  extracted  from 
the  blood.  Injected  into  an  animal,  it  produced  depression 
of  temperature,  followed  by  progressive  wasting,  but  no 
spasm  or  paralysis. 

Antitoxin. — If  an  animal  is  cautiously  injected  with 
tetanus  toxin,  commencing  the  treatment  with  a  weakened 
toxin,  and  increasing  the  dose  very  gradually,  a  high 
degree  of  immunity  is  ultimately  obtained,  and  the  blood- 
serum  acquires  marked  antitoxic  properties.  The  toxin 
is  obtained  by  growing  the  tetanus  bacillus  in  bouillon  in 
an  atmosphere  of  hydrogen  for  about  three  weeks,  and 
filtering  through  porous  porcelain.  To  obtain  an  active 
serum  treatment  has  to  be  prolonged,  a  horse  immunised 
by  the  writer  requiring  six  months.  The  antitoxic  serum 
so  obtained  is  by  far  the  most  active  of  any  of  the  sera, 
and  is  now  recognised  as  the  proper  remedy  to  use  in  cases 
of  tetanus  in  man.  The  antitoxic  treatment  of  tetanus 
is  not  nearly  so  successful  as  that  of  diphtheria,  and  for 
this  reason  :  in  diphtheria,  in  a  large  proportion  of  the 
cases,  a  local  manifestation  is  present  to  aid  diagnosis 
before  any  serious  absorption  of  the  toxin  has  taken  place, 
whereas  in  tetanus  the  disease  is  only  recognisable  by  the 
symptoms  induced  by  such  absorption.  Nevertheless, 
tetanus  antitoxin  should  always  be  employed  not  only 
in  the  fully  developed  disease,  but  also  in  certain  cases  as 
a  prophylactic.  As  the  toxin  is  at  once  fixed  by  the  nerve- 
tissue,  the  antitoxin  should  be  injected  into  the  central 
nervous  system  in  order  to  obtain  immediate  action. 


MALIGNANT  (EDEMA  425 

The  antitoxin  may  be  standardised  by  the  Roux  or  by 
the  Behring  method  (see  p.  280).  Recently  a  method 
analogous  to  that  used  for  standardising  diphtheria  anti- 
toxin has  been  introduced.1 


Clinical  Examination 

The  symptoms  of  tetanus  are  usually  so  obvious  that  a  bacterio- 
logical examination  is  not  needed  to  establish  the  diagnosis,  and 
unless  there  is  an  evident  wound  it  will  be  difficult,  if  not  impossible, 
to  detect  the  tetanus  bacillus. 

(1)  Prepare  several  smears  of  the  pus  or  discharge,  and  stain 
by    Gram's    method.     Examine    microscopically,    looking   for   the 
spore-bearing  rods  or  "  drum-sticks."     A  "  drum-stick  "  bacillus  is, 
however,  not  necessarily  the  tetanus  bacillus  (see  p.  420). 

(2)  If  "  drum-sticks  "  be  found,  an  attempt  may  be  made  to 
isolate  the  bacillus  by  making  anaerobic  plate  cultivations  from 
the  discharge,  after  heating  it  in  capillary  pipettes  to  80°  C.  for 
half  an  hour. 

(3)  Inoculate  mice  and  guinea-pigs  with  the  heated  discharge. 
If  they  die  with  tetanic  symptoms,  treat  the  pus  at  the  seat  of 
inoculation  as  in  (2). 


Malignant  (Edema 

Malignant  oedema  is  met  with  in  man  in  connection 
with  wounds  soiled  with  septic  matter,  compound  frac- 
tures, contused  and  lacerated  wounds,  etc.  Usually  there 
is  a  putrefactive  and  oedematous  condition  of  the  tissues 
with  subcutaneous  emphysema.  Animals  also  occasionally 
suffer  from  the  disease,  which  can  be  produced  artificially 
by  inoculation  with  dust,  dust  from  straw,  the  upper 
layers  of  garden  earth,  and  decomposing  animal  and 
vegetable  matter. 

If  a  guinea-pig  be  inoculated  subcutaneously  with  a 
little  garden  earth,  it  will  very  likely  die  in  forty- eight 

1  On  the  standardisation  and  therapeutic  use  of  tetanus  antitoxin, 
see  Hewlett's  Serum  Therapy,  1910. 


426  A  MANUAL  OF  BACTERIOLOGY 

hours.  Post  mortem,  the  subcutaneous  tissues  around 
the  seat  of  inoculation  will  be  found  to  be  cedematous  and 
blood-stained,  with  more  or  less  development  of  gas.  The 
internal  organs  are  only  slightly  altered,  but  the  spleen 
may  be  somewhat  enlarged.  The  juice  from  the  seat  of 
inoculation  will  be  found  to  contain  a  mixture  of  organisms, 
but  in  the  blood  and  organs  few  will  be  found.  Under  the 
capsule  of  the  spleen,  however,  long  slender  rods  may 
be  seen ;  these  are  the  bacilli  of  malignant  oedema. 

Morphology. — The  bacillus  of  malignant  oedema  is  a  long 
and  slender  rod,  several  of  which  may  be  united  into  a 
thread.  It  is  motile,  possesses  several  flagella,  and  is 
readily  stained  by  the  ordinary  anilin  dyes,  but  not  by 
Gram's  method.  It  spores  freely  at  temperatures  above 
20°  C.,  the  spores  being  large  and  central. 

Cultural  characters.- — The  bacillus  of  malignant  cedema 
is  strictly  anaerobic.  In  a  deep  stab  in  glucose-agar  it 
forms  a  thick  line  of  growth  in  the  needle  track,  with 
irregular  outline  and  greyish-white  in  colour.  There  is 
profuse  development  of  gas,  accompanied  by  a  foul  odour, 
and  attended  with  disruption  of  the  medium  into  several 
portions. 

The  bacillus  of  malignant  oedema  is  an  organism  which  has  to 
be  distinguished  from  anthrax,  and  there  should  be  no  difficulty  in 
doing  this.  Post  mortem,  the  spleen  is  rarely  found  much  enlarged 
in  malignant  oedema,  the  organism  is  not  very  abundant,  is  almost 
entirely  absent  from  the  blood,  and  is  only  found  under  the  capsule 
of  the  spleen,  not  at  its  centre.  If,  however,  several  hours  have 
elapsed  since  death  occurred,  the  organism  may  have  wandered 
into  the  blood  and  the  centre  of  the  spleen.  The  bacillus  of 
malignant  oedema  is  motile  under  anaerobic  conditions,  the  anthrax 
bacillus  non-motile  ;  the  former  occurs  as  a  long  slender  filament, 
which  on  staining  is  seen  to  consist  of  two  or  three  long  segments  ; 
it  does  not  stain  by  Gram's  method  (except  by  Claudius's  modifica- 
tion), and  is  strictly  anaerobic. 


EMPHYSEMATOUS  GANGRENE  427 


Bacillus  botulinus 

In  certain  forms  of  meat  poisoning  (see  Chap.  XXI)  van 
Ermengem  isolated  an  anaerobic  bacillus,  the  B.  botulinus.  It 
is  chiefly  met  with  in  ham  and  sausage,  and  the  symptoms  are 
caused  by  the  absorption  of  toxin,  which  has  a  special  effect  on  the 
nerve  centres. 

The  organism  is  a  large  Gram-positive  sporing  anaerobic  bacillus, 
often  occurring  in  pairs  or  in  short  chains.  In  glucose  gelatin  it 
forms  a  whitish  streak  in  the  line  of  the  stab,  with  lateral  out- 
growths, liquefaction  of  the  medium,  and  gas-formation.  The 
cultures  have  a  rancid  odour,  due  to  butyric  acid  production.  The 
colonies  in  gelatin  are  semi-transparent  spheres.  The  optimum 
growth  is  from  20°-30°  C.  The  source  of  the  organism  is  unknown, 
but  it  has  once  been  isolated  from  the  excreta  of  a  healthy  pig. 

The  B.  botulinus  in  broth  cultures  forms  a  potent  extra-cellular 
toxin,  which  is  toxic  both  by  injection  and  by  ingestion.  The  toxin 
is  also  produced  in  the  infected  ham,  sausage,  etc.  With  the  toxin 
an  antitoxin  can  be  prepared. 


Bacillus  Welchii  * 

Probable  synonyms. — B.  aerogenes  capsulatus  (Welch  and  Nuttall), 
Granulo -bacillus  saccliaro-butyricus  immobilis  liquefaciens  (Grass- 
berger  and  Schattenfroh),  B.  enteritidis  sporogenes  (Klein),  B.  per- 
fringens  (Veillon  and  Zuber),  gasphlegmon  bacillus  (Frankel). 
bacillus  of  acute  rheumatism  (Achalme  :  see  "  Rheumatism  "). 

This  organism  was  originally  described  by  Welch  and 
Nuttall  under  the  name  B.  aerogenes  capsulatus,  and 
occurs  in  conditions  accompanied  by  much  development 
of  gas  in  the  tissues,  as  in  cases  which  might  be  described 
either  as  phlegmonous  erysipelas  or  as  emphysematous 
gangrene,  especially  after  injuries.  It  is  also  met  with 

1  See  Welch  and  Nuttall,  Bull.  Johns  Hopkins  Hosp.,  vol.  iii,  1892, 
p.  81  ;  Welch,  '  Shattuck  Lecture,'  ibid.  vol.  xi,  1900,  p.  185  ;  Dunham, 
ibid.  vol.  viii.  1897,  p.  68  ;  Welch  and  Flexner,  Journ.  Exper.  Med., 
vol.  i,  1896,  p.  5  ;  Herter,  Bacterial  Infections  of  the  Digestive  Tract, 
1907  ;  Kamen,  Centr.  f.  Bakt.,  Orig.  xxxv,  1904,  pp.  554,  6S6  ;  Archiv. 
f.  Hyg.,  vol.  liii,  1905,  p.  128 ;  and  Blake  and  Lahey,  Journ.  Amer. 
Med.  Assoc.,  vol.  liv,  1910,  p.  1671. 


428  A  MANUAL  OF  BACTERIOLOGY 

occasionally  in  perforative  peritonitis  and  in  various 
septicaemic  and  pysemic  conditions,  in  the  puerperal  state,1 
complicated  stricture,  etc. 

The  B.  Welchii  is  widely  distributed,  and  has  been 
cultivated  from  the  soil,  dust,  and  contents  of  the  intestine. 
It  has  either  been  described  under  a  variety  of  names,  or 
a  group  of  closely  related  bacilli  may  exist.  Gas-bubbles 
found  in  the  blood  and  internal  organs  ("  foamy  organs  ") 
at  an  autopsy  seem  generally  to  be  due  to  this  organism, 
but  may  occasionally  perhaps  be  caused  by  other  putre- 
factive bacteria. 

Morphology. — The  B.  Welchii  is  a  non-motile,  sporing, 
anthrax-like  bacillus,  variable  in  size,  being  3  to  6  /x  in 
length  (Plate  XVII.  b).  It  occurs  singly,  in  short  chains, 
or  in  clumps,  and  occasionally  in  long  threads.  It  stains 
well  with  the  ordinary  anilin  dyes  and  also  by  Gram's 
method.  A  capsule  is  often  present,  but  spores  are  only 
formed  in  blood- serum  cultures. 

Cultural  characters. — The  B.  Welchii  grows  well  on  all 
the  ordinary  culture  media,  slowly  at  20°  C.,  rapidly  at 
blood-heat,  but  is  strictly  anaerobic.  It  forms  greyish- 
white  colonies  on  agar,  and  gelatin  is  liquefied.  In  glucose- 
broth  it  produces  at  first  a  diffuse  cloudiness,  but  later 
the  fluid  becomes  clear  and  a  whitish  viscid  sediment 
settles.  Milk  is  coagulated,  the  casein  forming  a  thick, 
stringy,  honeycombed  mass  on  the  surface  of  a  clear  watery 
whey.  On  potato  the  growth  is  almost  invisible.  There 
is  abundant  formation  of  gas  in  culture  media,  the  gas 
both  in  dextrose  media  and  in  milk,  according  to  Theobald 
Smith,  consisting  of  hydrogen  and  carbon  dioxide  in  the 
ratio  2  :  1  or  3  :  2. 

Pathogenicity. — The  B.  Welchii  is  pathogenic  for  guinea- 
pigs  and  mice,  but  slightly  so  for  rabbits.     The  whey  of  a 
milk  culture  in  quantities  of  0-5-2  c.c.  per  100  grm.  of 
1  See  Little,  Bull.  Johns  Hopkins  Hosp.,  vol.  xvi,  1905,  p.  136. 


PLATE  XVII. 


a.  Bacillus  tetani.     Film  preparation  of  a  pure  culture. 
X  1500. 


b.  Bacillus  Welchii.     Film  preparation  of  a  milk  culture. 
X  1000. 


BACILLUS  WELCHII  429 

body-weight  produces  death  in  a  guinea-pig  within  forty- 
eight  hours.  Post  mortem,  if  injected  subcutaneously, 
the  hair  strips  readily  from  the  skin,  which  may  be  green 
and  gangrenous  ;  the  subcutaneous  tissue  may  also  be 
green  and  gangrenous,  or  more  or  less  digested,  so  that  the 
skin  hangs  loose,  and  the  sac  formed  contains  gas  and 
exudation,  sometimes  scanty,  sometimes  abundant,  thin 
and  sanguinolent,  and  containing  numbers  of  bacilli. 
If  the  post-mortem  be  delayed,  or  if  the  heart-blood  be 
taken  up  into  tubes,  and  these  are  sealed  and  incubated 
for  some  hours,  many  of  the  bacilli  will  spore.  Pigeons, 
by  intra- muscular  inoculation,  are  also  susceptible.  Injected 
intravenously  into  a  rabbit,  the  animal  killed  immediately 
and  the  carcase  incubated  at  37°  C.  for  twenty-four  hours 
and  examined,  there  is  an  abundant  formation  of  gas, 
particularly  in  the  liver,  which  is  riddled  with  gas- 
bubbles.  This  is  a  very  characteristic  test  ( Welch- Nuttall 
test). 

The  B.  Chauvcei  also  produces  this  "  foaming  "  condition 
of  organs  when  similarly  treated,  but  spores  freely,  whereas 
the  B.  Welchii  does  not  spore  under  such  conditions. 
Monkeys  fed  with  considerable  numbers  of  B.  Welchii  are 
unaffected.  In  the  human  intestine  the  organism  is 
almost  absent  or  scanty  in  nurslings  and  children,  but 
becomes  more  and  more  abundant  as  age  advances.  It  is 
probable  that  it  is  capable  of  producing  necrotic  changes 
in  the  intestinal  mucous  membrane.  Different  strains 
seem  to  vary  much  in  virulence. 

Products  and  toxins. — The  gas  production  has  already 
been  mentioned.  Butyric  and  allied  acids  are  freely 
formed,  but  lactic  acid  is  scanty.  Indole  may  or  may  not 
be  produced.  Hsemolytic  substances  can  be  readily 
detected  in  blood-bouillon  cultures,  and  the  organism  is 
abundant  in  the  intestine  in  some  cases  of  primary  anaemia 
and  possibly  may  have  some  relation  to  the  condition. 


430  A  MANUAL  OF  BACTERIOLOGY 

In  some  cases  of  infection  the  blood- serum  agglutinates 
the  organism. 

Under  the  name  B.  enteritidis  sporogenes,  Klein  1  isolated  a  bacillus 
similar  to  the  B.  Welchii  from  the  evacuations  of  and  from  milk 
consumed  by,  patients  suffering  from  an  epidemic  diarrhoea  which 
occurred  in  St.  Bartholomew's  Hospital ;  as  did  Andrewes, 2  from 
cases  of  diarrhoea  admitted  into  the  same  hospital.  Klein  believed 
this  organism  to  be  the  cause  of  the  diarrhoea,  and  stated  that  it 
could  not  be  found  in  the  intestinal  evacuations  of  healthy 
individuals.  Klein  also  found  it  in  water,  sewage,  manure,  and 
milk.  The  writer,  however,  showed  that  it  could  generally  be 
found  in  the  normal  dejecta  also  in  road  and  laboratory  dust  and 
frequently  in  milk,  and  the  opinion  he  formed  was  that  it  was 
probably  a  ubiquitous  organism  and  had  little  to  do  with  the 
diarrhoea.3  Glynn  also  found  the  organism  to  be  very  widely 
distributed,  and  fed  guinea-pigs  with,  and  himself  ingested,  cultures 
without  result. 4 

The  B.  enteritidis  sporogenes  in  its  morphology,  staining  reaction, 
and  cultural  characters  is  almost,  if  not  quite,  identical  with  the 
preceding  organism,  the  B.  Welchii  or  B.  aerogenes  capsulatus  of 
Welch.  The  only  point  of  difference  between  them  is  that  the 
former,  according  to  Klein,  is  motile  and  flagellated,  while  the  latter, 
according  to  Welch,  is  non-motile  and  non-flagellated.  Spores  are 
only  formed  in  serum  or  gelatin,  not  on  agar.  It  is  abundantly 
present  in  sewage  and  sewage -contaminated  water  (see  Chap.  XXI). 

The  Clostridium  butyricum  of  Botkin,  an  energetic  butyric -acid- 
forming  anaerobic  bacillus  (p.  432),  produces  in  milk  changes 
similar  to  those  of  the  B.  Welchii,  but  is  non-pathogenic. 


Clinical    Examination    (Malignant    (Edema    and 
B.  Welchii) 

The  character  of  the  wound  and  discharge  will  probably  give 
some  indication  of  the  existence  of  infection  with  malignant  oedema 
or  with  B.  Welchii.  The  tissues  are  softened,  cedematous,  and  dis- 
coloured, and  soaked  with  a  foul-smelling,  sanguineous  fluid,  which 

1  Rep.  Ned.    Off.  Loc.  Gov.  Board,  1895-96,  p.  197  ;    ibid.  1897-98. 
p.  225. 

2  Ibid,  for  1896-97,  p.  225. 

3  Trans.  Jenner  Inst.  Prev.  Med.,  vol.  ii,  1899,  p.  70. 

4  Thomson  Yates  Lab.  Rep.,  vol.  iii,  Pt.  ii,  1901,  p.  131. 


BLACK  QUARTER  431 

may  be  frothy  from  the  development  of  gas.     Other  bacilli  will 
probably  be  present. 

(1)  Make  films  from  the  discharge.     Stain  some  with  Loffler's 
blue,  and  others  by  Gram's  method.     Examine  microscopically,  and 
look  for  bacilli  of  the  forms  described.     B.  Welchii  stains,  malignant 
oedema  does  not  stain,  by  Gram. 

(2)  Inoculate  two  guinea-pigs  subcutaneously  with  the  discharge 
or  with  portions  of  the  tissues.     It  the  animals  die,  look  for  the 
characteristic  organism. 

(3)  An  attempt  may  be  made  to  isolate  the  bacillus  by  anaerobic 
cultures    and    plate    cultivations,    prepared    from    unheated,    and 
heated  (80°  C.  for  ten  minutes),  material. 


Bacillus  cadaveris  sporogenes 

This  is  another  organism  isolated  by  Klein,1  and  has  to  be  dis- 
tinguished from  the  B.  Welchii.  The  two  organisms  are  morpho- 
logically very  similar  and  both  stain  by  Gram's  method,  but  the 
B.  cadaveris  sporogenes  does  not  produce  the  typical  changes  in 
milk.  In  a  culture  two  or  three  days  old  the  milk  below  the  cream 
layer  commences  to  clear,  and  later  this  change  proceeds  rapidly, 
so  that  at  the  end  of  a  week  three  layers  are  apparent — an  upper  of 
unchanged  cream,  a  middle,  yellowish  and  watery,  and  a  lower  of 
precipitated  casein.  Its  colonies  on  agar  are  also  different,  sending 
out  ramifying,  anastomosing  threads  from  their  margins,  and  it 
spores  freely  on  agar  in  two  to  three  days. 


Black  Quarter 

Syn.  :  Black  Leg,  Quarter  Evil,  Symptomatic  Anthrax,  Rausch- 
brand. 

Black  quarter  is  a  disease  affecting  sheep  and  oxen,  and  is  un- 
known in  man.  The  names  black  quarter,  black  leg,  and  quarter 
evil  are  derived  from  the  dark  discoloration  of  the  muscles  of  the 
leg  and  flanks  or  quarters  of  the  affected  animals.  When  the  muscles 
are  cut  into,  a  thin  sanguineous  fluid  exudes,  and  in  this  fluid  slender 
bacilli  are  present,  some  of  which  are  swollen  or  club-shaped  from 
the  presence  of  spores.  The  muscles  are  dark,  slightly  crepitant 
owing  to  the  presence  of  gas,  and  have  a  rancid  odour. 

The  organism,  the  B.  (Clostridium)  Chauvcei,  is  a  slender  rod 

1  Centr.f.  Bakt.  (lte  Abt.),  xxv,  p.  278. 


432  A  MANUAL  OF  BACTERIOLOGY 

never  forming  long  threads,  is  strictly  anaerobic  and  motile,  but 
loses  its  motility  in  the  presence  of  oxygen.  Some  of  the  rods  are 
cylindrical  throughout,  others  form  slender  spindles,  others  are  oval 
or  lemon-shaped.  It  stains  with  the  ordinary  anilin  dyes  but  not 
by  Gram's  method  (except  by  Claudius's  modification).  Occasion- 
ally in  the  tissues  it  seems  to  stain  by  Gram.  The  organism  forms 
endogenous  spores,  the  spore-bearing  rods  being  enlarged  or  club- 
shaped,  and  therefore  should  be  termed  a  "  clostridium." 

It  can  be  grown  in  deep  stabs  in  gelatin  and  agar.  Gelatin  is 
rapidly  liquefied.  In  glucose -agar  it  forms  a  thick,  irregular, 
greyish  growth,  with  much  development  of  foul-smelling  gas.  The 
writer  has  found  extreme  difficulty  in  isolating  and  in  maintaining 
cultures  of  the  organism.  The  guinea-pig  is  susceptible  if  inoculated 
subcutaneously  or  into  the  muscles,  the  bacilli  being  found  at  the 
seat  of  inoculation,  but  not  in  the  blood  or  internal  organs.  Artificial 
immunity  can  be  induced  in  various  ways  :  by  bacilli  attenuated 
by  heat  or  by  successive  cultivations,  or  by  heating  the  dried 
muscle  to  85°  to  90°  C.  for  six  hours  (Kitt),  also  by  inoculating  the 
susceptible  animal  at  the  tip  of  the  tail.  Hanna,1  by  growing  the 
organism  in  a  mixture  of  blood-plasma  and  broth,  obtained  toxins 
which,  by  careful  injection,  conferred  immunity  on  rabbits,  the 
animals  after  injection  yielding  an  antitoxic  serum. 

Hamilton  has  described  specific  anaerobic  bacilli  in  braxy, 
louping-ill,  and  other  diseases  of  sheep  and  deer.2 


Clostridium  butyricum 

An  anaerobic  organism  occurring  in  milk,  in  which  it  produces 
a  marked  butyric  acid  fermentation  with  changes  like  those  of  the 
B.  Welchii.  It  forms  short  rods,  and  also  long  ones  3  to  10  ^  in 
length,  and  filaments  are  met  with.  Spore -formation  takes  place 
freely  in  enlarged  segments.  It  forms  a  whitish  growth  on  agar, 
and  gelatin  is  rapidly  liquefied,  a  scum  forming  on  the  surface.  It 
is  non-pathogenic  (p.  430). 

1  Journ.  Path,  and  Bact.,  vol.  iv,  1897,  p.  383. 

2  Rep.    Louping-ill   and   Braxy    Com.,   Board    of    Agriculture    and 
Fisheries.  1906. 


CHAPTER  XIV 

ASIATIC  CHOLERA— SPIRILLUM  METCHNIKOVI— SPIRIL- 
LUM OF  FINKLER  AND  PRIOR— SPIRILLUM  TYRO- 
GENUM— SPIRILLUM  RUBRUM 

Asiatic  Cholera 

THE  bacteriological  study  of  Asiatic  cholera  may  be  said  to  date 
from  the  researches  of  Koch,  who  in  1884  was  sent  by  the  German 
Government  to  investigate  the  disease  in  Egypt  and  India.  He 
described  an  organism  present  in  the  intestine  and  in  the  dejecta 
which  he  believed  to  be  the  specific  contagium,  and  termed  it  the 
"  comma  bacillus  "  .from  its  curved  shape.  This  name  is  a  mis- 
leading one,  for  the  organism  is  not  shaped  like  a  printer's  comma, 
but  is  a  curved  rod  or  vibrio,  by  some  placed  in  the  genus  spirillum  ; 
however,  it  is  commonly  known  as  "  Koch's  comma  bacillus." 


Spirillum  (Vibrio)  choleras  asiaticae 

Morphology. — Curved  rods  with  rounded  ends  1  to  2  JUL 
in  length,  sometimes  forming  half  a  circle,  sometimes 
united  in  pairs  forming  an  S-shaped  curve  (Plate  XVII I.  a). 
It  is  present  in  the  intestine  and  in  the  alvine  discharges, 
especially  in  the  rice- like  flakes,  but  is  not  found  in  the 
blood,  organs,  or  tissues.  (Greig  has  twice  isolated  the 
organism  from  pneumonic  patches  in  the  lungs  and 
suggests  that  in  a  certain  percentage  of  cases  blood- 
infection  may  occur.)  In  the  rice-like  flakes  it  is  fre- 
quently so  numerous  that  in  a  film  the  "  commas  "  are 
arranged  in  "  ranks  and  files  "  parallel  to  one  another ; 
this  is  known  as  the  "  fish-in-stream  "  arrangement.  The 

433  28 


434  A  MANUAL  OF  BACTERIOLOGY 

vibrio  stains  well  with  the  ordinary  anilin  dyes,  especially 
with  dilute  carbol-fuchsin,  but  is  decolorised  by  Gram's 
method.  It  is  actively  motile,  and  typically  possesses  a 
single  terminal  flagellum  at  one  end  only,  but  there  is  some 
variation  in  this  respect.  Spores  are  not  formed,  though 
in  old  cultures  Hueppe  described  bodies  which  he  believes  to 
be  arthrospores.  In  such  cultures  the  bacilli  lose  their  regular 
shape,  and  swollen  and  distorted  involution  forms  are  seen. 

The  majority  of  the  organisms  in  a  young  agar  culture 
assume  the  vibrio  form,  but  in  broth  or  peptone  water 
cultures  two  or  three  days  old  they  are  longer  and  there 
is  a  tendency  for  them  to  become  somewhat  spirillar. 

Cultural  characters  and  biology. — The  Koch  vibrio  is 
aerobic  and  facultatively  anaerobic,  and  grows  well  on 
the  ordinary  culture  media  from  20°  to  37°  C.  It  grows 
readily  in  an  atmosphere  of  hydrogen,  but  does  not  develop 
in  one  of  carbonic  acid  gas. 

In  gelatin  plates  at  22°  C.  small  cream-coloured  colonies 
appear  in  about  twenty- four  hours,  soon  accompanied  by 
liquefaction,  so  that  in  two  or  three  days  the  plate  becomes 
pitted.  Microscopically,  the  young  colonies  are  rounded 
with  irregular  margins,  cream-coloured,  and  coarsely 
granular.  In  stab- cultures  development  occurs  all  along 
the  stab  as  a  whitish,  opaque,  punctate  growth,  thicker 
above  than  below.  Liquefaction  commences  about  the 
second  day  and  progresses  slowly ;  in  the  early  stage 
it  is  confined  to  the  surface,  and  looks  like  a  little  bead  or 
air-bubble  (Plate  XVIII.  6),  but  in  a  fortnight  or  so  the 
greater  part  of  the  gelatin  may  be  liquefied.  Liquefaction 
varies  greatly  both  in  rate  and  in  extent  in  different 
cultures  and  stocks ;  in  some  old  laboratory  cultures  it 
may  be  almost  absent.  On  surface  agar  a  thick,  moist, 
shining,  greyish  growth  quickly  develops  with  more  or  less 
crenated  margins,  often  becoming  brownish  when  old.  On 
blood-serum  much  the  same  growth  occurs  with  slow 


PLATE  XVIII. 


a.  Spirillum  chohrce.     Film  preparation  of  a  pure  culture. 
X  1500. 


6  c  d 

Gelatin  stab- cultures,  two  days  old,  of  (b)  Sp.  cholerce, 

(c)  Sp.  Metchnikovi,  (d)  Sp.  Finkleri, 


434  A  MANUAL  OF  BACTERIOLOGY 

vibrio  stains  well  with  the  ordinary  anilin  dyes,  especially 
with  dilute  carbol-fuchsin,  but  is  decolorised  by  Gram's 
method.  It  is  actively  motile,  and  typically  possesses  a 
single  terminal  nagellum  at  one  end  only,  but  there  is  some 
variation  in  this  respect.  Spores  are  not  formed,  though 
in  old  cultures  Hueppe  described  bodies  which  he  believes  to 
be  arthrospores.  In  such  cultures  the  bacilli  lose  their  regular 
shape,  and  swollen  and  distorted  involution  forms  are  seen. 

The  majority  of  the  organisms  in  a  young  agar  culture 
assume  the  vibrio  form,  but  in  broth  or  peptone  water 
cultures  two  or  three  days  old  they  are  longer  and  there 
is  a  tendency  for  them  to  become  somewhat  spirillar. 

Cultural  characters  and  biology. — The  Koch  vibrio  is 
aerobic  and  facultatively  anaerobic,  and  grows  well  on 
the  ordinary  culture  media  from  20°  to  37°  C.  It  grows 
readily  in  an  atmosphere  of  hydrogen,  but  does  not  develop 
in  one  of  carbonic  acid  gas. 

In  gelatin  plates  at  22°  C.  small  cream-coloured  colonies 
appear  in  about  twenty- four  hours,  soon  accompanied  by 
liquefaction,  so  that  in  two  or  three  days  the  plate  becomes 
pitted.  Microscopically,  the  young  colonies  are  rounded 
with  irregular  margins,  cream-coloured,  and  coarsely 
granular.  In  stab- cultures  development  occurs  all  along 
the  stab  as  a  whitish,  opaque,  punctate  growth,  thicker 
above  than  below.  Liquefaction  commences  about  the 
second  day  and  progresses  slowly ;  in  the  early  stage 
it  is  confined  to  the  surface,  and  looks  like  a  little  bead  or 
air-bubble  (Plate  XVIII.  b),  but  in  a  fortnight  or  so  the 
greater  part  of  the  gelatin  may  be  liquefied.  Liquefaction 
varies  greatly  both  in  rate  and  in  extent  in  different 
cultures  and  stocks  ;  in  some  old  laboratory  cultures  it 
may  be  almost  absent.  On  surface  agar  a  thick,  moist, 
shining,  greyish  growth  quickly  develops  with  more  or  less 
crenated  margins,  often  becoming  brownish  when  old.  On 
blood- serum  much  the  same  growth  occurs  with  slow 


PLATE  XVIII. 


a.  Spirillum  cholerce. 


Film  preparation  of  a  pure  culture. 
X  1500. 


6  c  d 

Gelatin  stab-cultures,  two  days  old,  of  (b)  Sp.  cholerce, 

{c)  Sp.  Metchnikovi,  (d)  Sp.  Finkleri, 


THE  COMMA  BACILLUS  435 

liquefaction.  A  thin  brownish  layer  is  formed  on  potato 
at  37°  C.  ;  and  broth  becomes  turbid,  a  delicate  film 
forming  on  the  surface.  Peptone  water,  or  Dunham's 
modification  of  it  (1  per  cent.  NaCl),  is  a  good  cultivating 
medium,  and  a  delicate  film  forms  on  the  surface.  In 
milk  it  multiplies  rapidly  without  curdling ;  neutral 
litmus  glucose- agar  is  reddened  from  the  development  of 
acid,  but  no  gas  is  produced  under  cultivation.  Acid, 
but  not  gas,  is  produced  from  glucose,  maltose,  saccharose, 
lactose,  and  starch. 

An  important  characteristic  of  the  cholera  vibrio  is 
the  rapid  formation  of  indole  in  considerable  quantity, 
and  the  reduction  of  nitrates  to  nitrites,  especially  in 
peptone  water.  This  forms  the  basis  of  the  important 
cholera-red  reaction  ;  a  few  drops  of  pure  sulphuric  or 
hydrochloric  acid  added  to  a  pep  tone- water  culture,  eight 
to  twelve  hours  old,  give  a  pink  colour,  and  the  colour  is 
intense  when  the  culture  is  two  to  three  days  old,  and  of 
a  purplish-red  colour,  like  that  of  potassium  permanganate. 
Some  specimens  of  "  peptone "  are  unsuitable  for  pre- 
paring the  peptone  water  used  for  obtaining  the  reaction, 
either  on  account  of  the  absence  of  a  tryptophane  nucleus, 
or  of  nitrates  and  nitrites.  The  medium  should  be  sugar- 
free,  and  the  addition  of  0-01  per  cent,  potassium  nitrate 
to  it  is  an  advantage.  Some  believe  that  two  pigments 
are  formed  in  the  reaction,  a  cholera- red  and  the  nitroso- 
indole  pigment.1  The  reducing  action  of  the  cholera 
vibrio  can  also  be  shown  by  growing  in  litmus  broth,  which 
becomes  decolorised  (Cahen's  test). 

Kraus  and  PrantschofI  2  noticed  that  certain  vibrios 
dissolved  red  blood- corpuscles,  but  came  to  the  conclusion 
that  no  true  recently  isolated  cholera  vibrio  is  hsemolytic 
(see  also  p.  441). 

1  Wherry,   Bureau  of  Government  Laboratories,   Manila,   Bulls.   19 
and  31,  1904  and  1905. 

2  Wien,  klin.  Woch.,  1906,  p.  299. 


J.50  A  MANUAL  OF  BACTERIOLOGY 

Strong,1  in  the  Philippines,  found  that  all  vibrios  which 
agglutinated  well  with  a  cholera  serum  were  genuine 
cholera  vibrios  and  that  none  of  them  was  haemolytic.  On 
the  other  hand,  Baerthlein  2  found  that  seven  freshly 
isolated  strains  of  the  cholera  vibrio  were  definitely  hsemo- 
lytic  in  suspensions  of  sheep's  corpuscles  in  from  twenty- 
four  to  forty-eight  hours.  Van  Loghem  3  employs  goat's 
blood  in  haemolytic  tests  for  the  cholera  vibrio.  He 
asserts  that  goat's  blood  is  quickly  haemolysed  by  haemo- 
lyshig  cholera-like  (e.g.  El  Tor,  p.  441)  vibrios,  but  that 
recently  isolated  cholera  strains,  if  they  haemolyse  at  all,  do 
not  do  so  for  some  time — twenty- four  to  forty- eight  hours. 

With  regard  to  this  important  question  of  haemolysis 
and  the  cholera  vibrios,  Van  Loghem  4  distinguishes  two 
types  of  blood  solution,  viz.  haemolysis  proper  and  haemo- 
digestion.  He  asserts  that  the  apparent  haemolysis  on 
a  blood- agar  plate  occasionally  occurring  with  the  true 
cholera  vibrio  is  really  haemo- digestion.  He  distinguishes 
the  two  conditions  by  the  tint  of  the  haemolytic  zone — 
red  in  true  haemolysis  and  greenish  in  haemo- digestion — 
and  spectroscopically  the  affected  zone  shows  oxyhae- 
moglobin  in  haemolysis  but  not  in  haemo-digestion.  The 
blood  agar  used  for  the  plates  is  composed  of  ordinary 
nutrient  agar  with  an  addition  of  11-12  per  cent,  of 
defibrinated  goat's  blood. 

The  cholera  vibrio  retains  its  vitality  in  cultures  for  a 
month.  It  can  multiply  in  water  and  on  the  surface  of 
moist  linen,  but  rapidly  dies  on  drying.  Its  thermal 
death-point,  according  to  Sternberg,  is  52°  C.  with  an 
exposure  of  four  minutes.;  according  to  Kitasato,  55°  C. 
in  about  ten  minutes.  '  It  is  easily  destroyed  by  the 
ordinary  germicides. 

1  Philippine  Journ.  of  Science,  vol.  v,  1910,  p.  403. 

2  Arb.  aus  dem  kaiserl.  Gesundheitsamte,  xxxvi,  1911. 

3  Centr.f.  Bakt.,  Abt.  I  (Originate),  Ivii,  1911,  p.    289. 

4  Ibid.  Ixx,  1913,  p.  70. 


SURVIVAL  OF  THE  COMMA  BACILLUS      437 

In  some  experiments  by  Dempster1  it  was  found  that 
the  comma  bacillus  lived  from  three  to  five  days  in  dry 
soil,  but  only  one  day  in  an  artificially  dried  soil,  while 
in  moist  soil  it  lived  from  twenty- eight  to  sixty-eight  days. 
In  peat,  however,  it  was  invariably  dead  within  twenty- 
four  hours.  In  sterilised  salt  solution  (0-75  per  cent.)  the 
comma  bacilli  were  alive  on  the  159th  day,  and  in  fresh 
urine  (sterilised)  they  lived  fourteen  days  at  37°  C.  and 
twenty-nine  days  at  22°  C. 

In  sterilised  distilled  water  the  cholera  vibrio  usually 
rapidly  dies,  as  a  rule  within  twenty-four  hours.  The 
addition  of  sodium  chloride  greatly  increases  the  length  of 
time  it  may  remain  alive,  a  survival  of  five  or  six  weeks 
having  been  recorded.  In  ordinary  sterilised  potable 
waters  it  may  survive  many  months.  In  unsterilised 
potable  waters  its  survival  is  greatly  influenced  by  the 
presence  of  salts  ;  in  some  cases  it  dies  out  rapidly ;  in 
others,  especially  in  those  containing  a  large  proportion 
of  salts,  it  may  remain  alive  for  some  time.  Houston  2 
found  that  cholera  vibrios  die  very  rapidly  in  raw  Thames, 
Lee,  and  New  River  waters  as  the  result  of  storage  in  the 
laboratory.  At  least  99-9  per  cent,  perish  within  one 
week,  and  it  was  not  possible  to  isolate  any,  even  from 
100  c.c.  of  the  water,  three  weeks  after  infection.  Klein  3 
found  that  the  cholera  vibrio  could  retain  its  vitality  for 
at  least  fourteen  days  in  unsterilised  sea- water,  while 
from  the  interior  of  oysters,  kept  in  water  infected  with 
the  vibrios,  it  was  obtained  up  to  nine  days  after  infection. 
In  sterilised  sewage  the  cholera  vibrio  multiplies  and 
survives  for  months  ;  in  unsterilised  sewage  it  may  survive 
for  two  to  four  weeks  (Houston). 

Pathogenicity. — The  disease  is  spread  mainly  by  infected 

1  Med.-Chir.  Trans.,  vol.  Ixxvii,  1894,  p.  263. 

2  Metropolitan  Water  Board,  Fifth  Rep.  on  Research  u-o)k,  1910. 

3  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1896,  p.  135. 


438  A  MANUAL  OF  BACTERIOLOGY 

water ;  milk,  salads,  vegetables  and  flies  are  other 
sources  of  infection.  The  organism  has  been  found  in 
the  dejecta  of  contacts  not  suffering  from  the  disease,  and 
it  may  sometimes  persist  for  long  periods  after  convales- 
cence. In  these  cases  the  vibrio  may  sometimes  be  located 
in  the  biliary  tract.  Crendiropoulo  examined  the  stools 
of  34,461  persons  on  ships  coming  from  cholera-infected 
ports.  Cultures  of  vibrios  were  obtained  from  63  of  these, 
of  which  23  were  agglutinated,  and  40  were  not  agglu- 
tinated, by  a  high-titre  cholera  serum. 

The  relation  of  the  cholera  vibrio  to  the  disease  has 
been  a  very  vexed  question  in  the  past,  but  the  outcome 
of  the  voluminous  researches  which  have  been  made  is 
to  confirm  Koch's  work.  The  organism  is  found  in  all 
cases  of  cholera,  and  several  instances  of  laboratory 
infection  from  cultures  have  been  recorded. 

None  of  the  lower  animals  suffers  from  or  contracts  a 
disease  in  any  way  comparable  to  Asiatic  cholera,  so  that 
the  test  of  animal  experiments  cannot  be  applied  except 
in  the  case  of  young  suckling  rabbits  (see  below,  "  Anti- 
serum  ").  By  first  neutralising  the  acidity  of  the  gastric 
juice  by  an  injection  of  sodium  carbonate  solution  into 
the  stomach,  then  diminishing  peristalsis  by  an  injection 
of  tincture  of  opium  into  the  peritoneal  cavity,  and  finally 
injecting  a  broth  culture  of  the  cholera  vibrio  into  the 
stomach,  Koch  succeeded  in  inducing  in  guinea-pigs  a 
condition  somewhat  similar  to  cholera  in  man — namely, 
indisposition  with  falling  temperature,  weakness  of  the 
extremities,  and  death  in  forty-eight  hours.  Post  mortem, 
the  small  intestine  was  congested  and  filled  with  a  watery 
fluid  containing  large  numbers  of  the  vibrios.  Injected 
into  the  peritoneal  cavity  of  mice,  guinea-pigs  and  rabbits, 
the  vibrio  produces  death  from  a  general  septicffimia,  and 
intra-muscular  inoculation  into  pigeons  is  sometimes  fatal. 
The  virulence  varies  much  and  is  lost  under  cultivation. 


OCCURRENCE  OF  VIBRIOS  439 

Metchnikoff  x  ascribes  the  immunity  of  animals  to  intes- 
tinal cholera  as  largely  due  to  the  inhibitory  action  of 
the  other  organisms  present  in  the  digestive  tract.  In 
man  digestive  disturbances  are  often  an  important  pre- 
disposing cause  of  an  attack.  The  acidity  of  the  gastric 
juice  is  also  probably  a  means  of  defence  (see  "  Water  "). 

The  blood-serum  of  an  animal  immunised  by  injections 
of  the  cholera  vibrio  gives  a  typical  agglutination  reaction 
with  recent  cultures  of  the  organism.  The  reaction  can 
also  be  obtained  with  the  blood-serum  of  cholera  patients, 
sometimes  as  early  as  the  first  day  of  the  disease,  but  it  is 
usually  of  little  use  for  diagnostic  purposes,  as  the  disease 
generally  runs  such  a  rapid  course. 

Occurrence  of  the  vibrio. — That  the  cholera  vibrio  is 
etiologically  associated  with  the  disease  seems  to  be 
beyond  any  doubt,  and  so  constant  is  its  presence  in  true 
cholera  that  all  investigators,  even  those  who  at  one  time 
opposed  Koch's  views,  rely  on  its  detection  for  the  bac- 
teriological diagnosis.  The  matter,  however,  has  become 
complicated  owing  to  the  detection  in  various  natural 
waters  of  pathogenic  vibrios  which,  although  not  identical 
with  the  cholera  vibrio  of  Koch,  resemble  it  so  closely  that 
it  is  difficult  to  classify  them  as  anything  but  varieties 
of  the  cholera  vibrio.  In  certain  epidemics  in  India  varia- 
tions have  also  been  noted  in  the  cholera  vibrios  that  have 
been  isolated.  Sanarelli 2  isolated  from  the  Seine  and 
Marne  thirty-two  vibrios,  of  which  four  were  almost  indis- 
tinguishable from  cholera,  except  that  they  were  only 
slightly  pathogenic,  but  by  passage  through  a  series  of 
animals  their  pathogenic  power  was  much  enhanced. 
Sanarelli  believed  that  these  were  the  descendants  of  true 
cholera  vibrios  that  had  gained  access  to  the  rivers  during 
some  previous  epidemic  of  cholera.  At  the  same  time  it  is 

1  Ann.  dc  I Inst.  Pasteur,  vii,  pp.  403,  562  ;   vol.  viii,  pp.  257,  520. 

2  Ibid,  vii,  p.  693,  and  ix,  p.  129. 


440  A  MANUAL  OF  BACTERIOLOGY 

to  be  noted  that  vibrios  may  also  be  present  in  the  normal 
intestinal  tract  of  man  and  animals,  and  may  therefore 
gain  access  to  streams  (Sanarelli).  D unbar  similarly,  from 
the  Elbe,  Rhine,  and  other  rivers,  isolated  a  number  of 
vibrios  which  could  not  be  distinguished  from  the  cholera 
vibrios  (Spirillum  Elwers).  It  was  afterwards  noticed  that 
some  of  these  under  certain  conditions  of  oxidation  and 
temperature  became  phosphorescent,1  but  Rumpel  2  has 
also  found  that  cultures  of  the  genuine  cholera  vibrio  may 
exhibit  phosphorescence,  so  this  cannot  be  used  as  a 
differential  character  for  the  separation  of  non- choleraic 
forms.  Neisser  isolated  a  vibrio,  which  he  termed  Vibrio 
Berolinensis,  which  agreed  with  the  cholera  vibrio  in  every 
particular  except  that  the  colonies  in  a  gelatin  plate  in 
forty-eight  hours  were  invisible  to  the  naked  eye.  Heider 
found  in  the  Danube  a  spirillum,  named  by  him  the  Vibrio 
Danubicus,  which  resembled  the  cholera  vibrio  closely, 
but  its  colonies  were  somewhat  different,  and  it  was  more 
actively  pathogenic  to  mice.  Ivanoff  similarly  obtained  a 
vibrio  which  could  only  be  distinguished  from  cholera  by 
the  finer  granulation  of  its  colonies  and  more  distinct 
spiral  form.  Lastly,  there  is  the  Vibrio  Massowah,  isolated 
from  an  epidemic  of  cholera  at  Massowah,  which  differs 
from  the  Koch  vibrio  in  having  two  terminal  flagella  at 
each  end.  Cunningham  has  also  described  several  vibrios 
differing  but  slightly  from  the  cholera  vibrio. 

Applying  the  Pfeiffer  and  agglutination  tests  to  the 
vibrios  in  question,  the  following  results  were  obtained. 
In  the  first  place,  each  of  the  organisms  gives  a  complete 
positive  reaction  to  both  tests  with  its  own  serum  ;  this, 
of  course,  is  only  to  be  expected.  Pfeiffer  found  that, 
using  his  reaction,  the  variety  Ivanoff  gave  a  positive 
reaction  with  cholera  serum,  and  Durham  found  that 

1  Centr.f.  Bakt.  (ltc  Abt.),  xviii,  1895,  p.  424  (Kutscher). 

2  Munch,  med.  Wochensch).,  1895,  No.  3. 


EL  TOR  VIBRIOS  441 

Ivanoff  and  Berolinensis  reacted  completely  with  cholera 
serum.  Conversely,  positive  reactions  with  cholera  vibrios 
were  obtained  with  Massoivah,  Danubicus,  and  Elwers  sera, 
while  Massowah  and  Elwers  react  completely  to  each  other. 
From  these  considerations  it  would  therefore  seem  probable 
that  some  of  these  vibrios — Sanarelli,  Berolinensis,  and 
Ivanoff — may  be  varieties  of  the  Koch  vibrio.  The  Massowah 
vibrio  is  usually  considered  not  to  be  a  true  cholera  vibrio. 

Ruffer  1  in  1905  at  El  Tor  isolated  vibrios,  which  may 
be  distinguished  as  "El  Tor  vibrios,"  from  the  intestine 
of  pilgrims  returning  from  Mecca  and  suffering  from  various 
diseases  (dysentery,  diarrhoea,  pneumonia,  rheumatism), 
but  among  whom  there  had  been  no  cholera,  and  who  had 
not  been  in  contact  with  cholera.  These  vibrios  were  sub- 
j  ected  to  detailed  examination  by  the  agglutination,  satura- 
tion and  fixation  tests,  and  Pfeiffer's  reaction  with  Berlin 
cholera-immune  serum,  and  also  by  the  haemolysis  test. 
Vibrios  isolated  from  a  previous  epidemic  of  cholera  (re- 
ferred to  as  Group  1),  and  other  vibrios  isolated  from  cholera 
and  other  stool  (Groups  3  and  4),  were  also  compared 
with  the  El  Tor  vibrios.  RufTer's  results  were  as  follows  : 

Group  1  (undoubted  cholera  vibrios). — Those  which 
react  positively  to  the  four  principal  tests  with  cholera 
serum — namely,  the  agglutination,  saturation,  and  fixation 
tests,  and  Pfeiffer's  reaction.  They  do  not  haemolyse, 
even  when  remaining  in  contact  with  red  corpuscles  for 
three  days  at  the  temperature  of  the  laboratory. 

Group  2. — The  second  group  contains  the  vibrios  agglu- 
tinated by,  and  giving  the  saturation  and  PfeifTer's  reactions 
with,  cholera  serum,  but  not  fixing  the  cholera-immune 
body.  These  vibrios  are  strongly  hsemolytic.  This  group 
consists  of  the  El  Tor  vibrios  only. 

1  Researches  on  the  Bacteriological  Diagnosis  of  Cholera.  Sanitary, 
Maritime,  and  Quarantine  Council  of  Egypt,  Alexandria,  1907.  (Also 
Brit.  Med.  Journ.,  1907,  vol.  i,  p.  735.) 


442  A  MANUAL  OF  BACTERIOLOGY 

Group  3. — The  third  group  is  formed  by  vibrios  which 
are  not  agglutinated  by  immune  serum,  nor  give  the 
saturation  or  Pfeiffer's  reaction,  but  fix  the  cholera-immune 
body.  These  vibrios  also  hsemolyse,  but  feebly  and  late, 
often  only  after  thirty-six  to  forty-eight  hours. 

Group  4. — The  last  group  is  formed  by  strongly  hsemo- 
lytic  vibrios  not  reacting  at  all  to  cholera-immune  serum. 

Buffer  concludes  that  the  El  Tor  vibrios  are  not  genuine 
cholera  vibrios.  He  says  :  "  The  only  possible  classifica- 
tion is  to  group  together  all  the  vibrios  reacting  in  the 
same  way  to  all  tests,  separating  them  from  those  which, 
under  the  same  conditions,  behave  in  a  different  way.  If 
this  method  be  applied  to  the  vibrios  found  at  El  Tor, 
there  is  no  difficulty  in  distinguishing  them  from  the  true 
cholera  vibrios,  in  spite  of  several  of  the  reactions  of  both 
being  similar.  And  it  follows  also  that  the  agglutination, 
saturation  and  Pfeiffer's  tests  are  not  in  themselves  of 
absolute  diagnostic  value  for  cholera  vibrios." 

Neufield  and  Haendel,1  however,  after  a  re- examination 
of  some  of  these  vibrios,  consider  that  they  are  true  cholera 
vibrios.  The  matter  therefore  remains  undecided. 

Klein  found  that  the  cholera  vibrio  kept  in  sea- water 
showed  marked  variation  from  the  original  strain.  In 
the  East  many  cases  of  cholera  are  mixed  "  vibrionic  " 
infections  ;  the  stools  may  contain  several  varieties  of 
vibrios,  some  agglutinating  with  cholera  serum,  others 
not ;  some  monociliate,  others  multiciliate. 

It  may  be  that,  like  the  B.  dysenteries,  the  cholera  vibrio 
is  not  a  single  definite  organism,  but  that  cholera  may  be 
caused  by  any  one  of  a  group  of  closely  allied  vibrios. 

Toxins. — Brieger  in  1887  obtained  cadaverin  and  pu- 
trescin  and  two  other  basic  bodies  from  cholera  cultures. 
Brieger  and  Frankel  isolated  a  tox-albumin,  and  Gamaleia 
a  ferment-like  body.  Hueppe  believes  that  the  cholera 

1  Arbeit,  a.  d.  Kais.  Gesundheitsamte,  xxvi,  1907,  p.  536. 


CHOLERA  TOXINS  443 

poison  is  a  tox- albumin  formed  in  the  culture  medium, 
but  that  immunising  substances  are  derived  from  the 
bacterial  cells. 

Rontaler  compared  the  chemical  products  of  the  ordinary 
cholera  and  of  the  Massowah  spirilla,  and  could  find  little 
difference  between  them. 

Wesbrook  1  obtained  albumoses  and  other  bodies  from 
alkali-albumin,  egg,  and  Uschinsky  medium,  cultures. 
This  observer  also  found  aerobic  cultures  of  the  cholera 
vibrio  to  be  much  more  toxic  than  anaerobic  ones. 

Pfeiffer  found  that  cholera  cultures  killed  with  chloro- 
.form  vapour  contained  a  toxic  substance  fatal  to  guinea- 
pigs  in  small  doses,  with  extreme  collapse.  He  believed 
the  substance  to  be  an  integral  part  of  the  bacterial  cells. 

Metchnikoff,2  Roux  and  Salimbeni  demonstrated  the 
existence  of  a  soluble  cholera-poison  in  a  very  ingenious 
manner.  Collodion  sacs  of  2  c.c.  to  3  c.c.  capacity  were 
sterilised,  filled  with  peptone  solution,  inoculated  with 
the  cholera  spirillum,  and  closed.  The  closed  sac  was 
then  introduced  into  the  peritoneal  cavity  of  a  guinea-pig, 
which  died  in  three  or  four  days  from  the  effects  of  the 
soluble  toxins  dialysing  through  the  walls  of  the  sac  (see 
also  next  page). 

Brau  and  Dernier  3  obtained  a  toxic  filtrate  by  culti- 
vating the  cholera  vibrio  in  a  medium  consisting  of  horse- 
serum  with  an  addition  of  10  per  cent,  of  defibrinated 
horse-blood. 

Macfadyen  obtained  a  highly  toxic  endotoxin  by  tritura- 
ting cholera  cultures  with  liquid  air.4 

Emmerich  5  strongly  supports  the  view  that  the  cholera 
intoxication  is  not  a  toxin  intoxication,  but  is  due  to 

1  Journ.  of  Path,  and  Bact.,  vol.  iv,  1896,  p.  1. 

2  Ann.  de  VInst.  Pasteur,  x,  1896,  p.  257. 

3  Ibid,  xx,  1906. 

4  Lancet,  1906,  vol.  ii,  p.  494. 

6  Munch,  med.  Wochenschr.,  1911,  No.  18,  p.  942. 


444  A  MANUAL  OF  BACTERIOLOGY 

nitrite  poisoning,  the  nitrites  being  produced  by  the 
reducing  action  of  the  vibrios  on  nitrates  present. 

Anti-serum. — By  growing  the  cholera  vibrio  in  a 
shallow  layer  with  free  access  of  oxygen  in  a  peptone 
gelatin-salt  medium,  Metchnikoff  and  his  co-workers 
obtained  a  toxic  fluid  after  three  or  four  days  growth. 
During  incubation  the  fluid  becomes  concentrated  to  about 
one-eighth  by  evaporation.  After  filtration,  0-25  c.c. 
killed  a  300-grm.  guinea-pig  in  eighteen  hours.  Goats, 
inoculated  with  increasing  doses  of  this  toxin,  com- 
mencing with  10  c.c.  and  reaching  200  c.c.  in  six  months, 
become  immunised  and  yield  an  antitoxic  serum,  1  c.c. 
of  which  will  neutralise  four  times  the  lethal  dose  of  toxin. 
Metchnikoff  had  previously  found  that  young  suckling 
rabbits  suffer  from  an  intestinal  cholera  when  fed  with 
cultures,  so  that  the  effect  of  the  cholera  antitoxin  in 
preventing  intestinal  cholera  could  be  tested  on  these 
animals.  Experiment  showed  that  of  the  treated  rabbits, 
51  per  cent,  survived,  of  the  untreated  only  19  per  cent. 
Salimbeni  employed  a  serum  prepared  in  this  manner  in 
the  treatment  of  cases  of  cholera  in  the  Russian  epidemic, 
1910. 

Animals  may  be  inoculated  with  dead  and  living  cultures 
and  an  immune  serum  so  prepared,  but  no  practical  value 
has  yet  attended  the  use  of  anti-sera  in  the  treatment  of 
cholera.  Macfadyen  immunised  a  goat  with  cholera- cell 
juice,  and  obtained  a  serum  of  which  5 -J-^-  c.c.  protected  a 
guinea-pig  against  three  lethal  doses  of  cholera  culture. 

The  writer  prepared  an  anti-endotoxic  serum  in  this 
manner,  with  which  a  few  cases  of  cholera  were  treated 
in  Russia.1 

Vaccine. — Ferran  in  1885  first  prepared  a  vaccine  by 
making  cultures  (mixed)  in  broth  from  cholera  stools  and 
injecting  0-3-0-5  c.c.  subcutaneously,  but  the  reports  of 

1  Lancet,  1910,  vol.  ii,  October  22. 


CHOLERA  VACCINE  445 

the  commissions  sent  to  investigate  the  method  were 
unfavourable. 

Haffkine  subsequently  prepared  a  vaccine  against 
cholera  from  cultures  of  the  Koch  vibrio,  which  seems 
to  be  efficacious  in  preventing  the  disease.  For  example, 
a  number  of  labourers  were  inoculated  during  an  epidemic, 
and  among  the  inoculated  the  mortality  was  only  2-25, 
whereas  among  the  uninoculated  it  was  nearly  19  per  cent. 
In  another  instance  amongst  654  uninoculated  there  were 
71  deaths,  a  mortality  of  10-86  per  cent.,  while  among 
402  inoculated  there  were  only  12  deaths,  a  mortality  of 
2-99  per  cent.,  and  a  reduction  in  mortality  of  72-47  per 
cent. 

In  the  Haffkine  method  two  vaccines  are  made  use  of. 
The  first  or  weak  vaccine  is  prepared  from  cultures  of  the 
cholera  vibrio  attenuated  by  growing  on  the  surface  of 
agar,  with  free  aeration,  for  several  generations.  The 
second  or  strong  vaccine  is  prepared  by  enhancing  the 
virulence  of  a  cholera  culture  by  a  succession  of  passages 
through  the  peritoneal  cavity  of  guinea-pigs.  The  viru- 
lence of  this  culture  must  be  maintained  in  the  same 
manner. 

For  making  both  vaccines,  "  standard  "  agar  cultures 
are  employed.  These  are  tubes  in  which  the  sloping 
surface  of  agar  measures  15  cm.  in  length,  and  the  cultures 
are  incubated  for  twenty-four  hours.  The  whole  growth 
on  such  a  tube  is  emulsified  in  8  c.c.  of  broth  or  salt  solu- 
tion ;  the  dose  of  this  is  1  c.c.,  and  the  living  vaccines  are 
injected  into  the  flank,  the  second  or  strong  being  given 
seven  to  ten  days  after  the  first  or  weak.  Haffkine  1  in 
a  recent  study  on  cholera  inoculation  suggests  the  use 
of  the  strong  vaccine  "  devitalised."  The  devitalised 
vaccine  may  be  prepared  by  two  methods,  (a)  prolonged 
cultivation  in  broth  and  treatment  of  the  culture  with 

1  Preventive  Inoculation  against  Cholera  (W.  Thackcr  &  Co.,  1913). 


446  A  MANUAL  OF  BACTERIOLOGY 

heat  and  carbolic  acid,  (6)  cultivation  on  agar  and  treat- 
ment with  carbolic  acid. 

Besredka  l  claims  that  an  immediate  and  lasting  (six 
months)  immunity  may  be  produced  by  making  a  mixture 
of  cholera  culture  and  cholera-immune  serum,  allowing  this 
to  stand  for  twelve  hours,  heating  to  56°  C.  for  one  hour 
and  then  injecting  subcutaneously. 

Strong  2  prepares  a  vaccine  from  autolysed  cultures. 
The  cholera  vibrio  is  grown  on  surface  agar  for  twenty- 
four  hours  at  37°  C.  ;  the  growth  is  then  washed  off  with 
sterile  water,  the  suspension  is  kept  at  60°  C.  for  twenty- 
four  hours,  and  then  at  37°  C.  for  two  to  five  days,  and  is 
finally  filtered  through  a  porcelain  filter. 

Clinical  Diagnosis 

Some  of  the  rice-like  flakes  should  be  picked  out  of  the  stool  and 
well  rinsed  in  sterile  salt  solution. 

1.  From  one  of  the  whitish,  slimy,  rice-like  flakes  in  the  evacua- 
tions or  the  intestine  films  are  prepared,  stained  with  Loffler's  blue, 
washed,  dried,  and  mounted.     If  on  examination  large  numbers  of 
curved  rods  lying  in  groups  parallel  to  one  another  are  observed, 
the  diagnosis  of  Asiatic  cholera  may  be  made  with  some  degree  of 
certainty.     Koch  states  that  this  is  so  in  quite  half  the  cases, 
especially  the  acute  ones.     (Single,  or  a  few,  vibrios  are  of  no 
diagnostic  significance  ;   they  may  occur  in  normal  and  diarrhrea 
stools.     The  presence  of  numbers  of  vibrios  having  the  "  fish-in  - 
stream  "  arrangement  is  also  not  absolutely  characteristic.) 

2.  Gelatin  and  agar  plates  should  be  prepared  from  an  emulsion 
of  rice-like  flakes.     Agar  plates  are  best  prepared  by  smearing  the 
flake  over  the  surface  of  the  solidified  agar.     The  plates  are  incubated 
at  22°  C.  and  37°  C.  respectively.     In  the  gelatin  plates  the  charac- 
teristic colonies  of  the  cholera  vibrios  should  be  recognisable  in 
about  twenty-four  hours,  in  the  agar  plates  in  from  twelve  to 
sixteen  hours.     The  likely  colonies  should  be  examined  microsco- 
pically and  peptone-water  and  other  cultures  prepared  from  them. 

A  better  medium  to  employ  is  Dieudonne's  blood  alkali  agar. 

1  Ann.  de  VInst.  Pasteur,  1902,  p.  918. 

2  Bureau  of  Gov.  Laboratories,  Manila,  Bull.  No,  16,  1904  (Bibliog.). 


SPIRILLUM  METCHNIKOVI  447 

Equal  parts  of  defibrinated  ox-blood  and  normal  caustic  potash 
solution  are  mixed  and  sterilised  in  the  steamer.  Of  this  30  c.c.  are 
mixed  with  70  c.c.  of  3  per  cent,  peptone-agar  (neutral  to  litmus), 
previously  melted.  Plates  are  poured  and  kept  at  60°  C.  for  half 
an  hour,  and  are  then  allowed  to  stand  for  twenty -four  hours  for 
ammonia  to  evaporate.  On  this  medium  few  organisms  except  the 
cholera  vibrio  develop  (but  cholera-like  vibrios  develop  equally 
well). 

3.  With   other   rice-like    flakes   several   peptone-water   cultures 
should   be  prepared  and  incubated  at  37°  C.     This  is  best  done  in 
the  small  Erlenmeyer  flasks  containing  a  shallow  layer  (1-2  cm. 
deep)  of  Dunham's  peptone -water,  without  wool  plugs,  but  capped 
with  a  piece  of  sterile  filter-paper.     In  eight  to  ten  hours  the  upper 
layers  of  the  fluid  should  be  examined  microscopically  for  the 
presence  of  vibrios,  and  gelatin,  agar  or  Dieudonne  agar  plates  and 
subcultures  in  peptone-water  are  also  made  by  inoculating  from  the 
surface  layer  of  fluid.     The  peptone -water  culture  may  then  be 
tested  for  the  presence  of  indole  by  carefully  adding  a  few  drops 
of  pure  concentrated  sulphuric  acid.     In  cases  of  Asiatic  cholera 
the  indole  reaction  can  be  obtained  as  early  as  eight  hours  after 
inoculation. 

If  vibrios  are  found  in  the  peptone -water  or  other  cultures,  they 
should  be  tested  for  agglutination  with  a  high-titre  cholera-immune 
serum  ;  if  positive  results  are  obtained,  the  diagnosis  is  practically 
certain.  The  haemolysis  test  should  also  be  applied,  as  it  is  com- 
paratively simple  (p.  182). 

4.  The  saturation  and  fixation  tests  and  Pfeiffer's  reaction  may 
also  be  applied. 

5.  If  the  case  has  lasted  some  time,  the  agglutination  reaction 
may  be  applied,  testing  the  patient's  serum  on  a  known  strain  of 
cholera  vibrio,  but  this  is  of  doubtful  value. 


Spirillum  Metchnikovi 

Isolated  by  Gamaleia  from  the  intestinal  contents  of  chickens 
dead  of  an  infectious  gastro -enteritis  which  occurred  in  certain  parts 
of  Russia.  The  disease,  although  resembling  chicken  cholera  in 
some  respects,  is  quite  distinct  from  the  latter.  This  spirillum  forms 
curved  rods  and  spiral  filaments,  generally  slightly  shorter,  thicker 
and  more  curved  than  the  Koch  vibrio.  It  is  decolorised  by 
Gram's  method,  and  is  best  stained  with  weak  carbol-fuchsin.  It  is 
readily  cultivated,  and  is  aerobic  and  facultatively  anaerobic.  In 


448  A  MANUAL  OF  BACTERIOLOGY 

gelatin  plates  it  forms  small  whitish  colonies,  visible  within  twenty 
hours,  which  grow  more  rapidly  than  the  cholera  vibrio,  and  in 
two  or  three  days  produce  marked  areas  of  liquefaction.  In  a 
stab-culture  in  gelatin  a  whitish  granular  growth  occurs  along  the 
line  of  puncture  with  liquefaction,  much  like  that  of  the  Koch 
vibrio,  but  the  rate  of  growth  and  the  liquefaction  are  more 
rapid  (Plate  XVIII.  c).  Grown  in  eggs  by  Hueppe's  method 
typical  appearances  are  produced.  After  ten  days  the  white  becomes 
transformed  into  a  yellowish  limpid  liquid,  while  the  yolk,  though 
retaining  its  form  and  consistence,  is  quite  black.  On  surface  agar 
a  thick  cream-coloured  layer  develops  ;  on  potato  the  growth  is 
brownish,  and  milk  is  coagulated.  It  grows  freely  in  broth  and 
peptone-water,  the  fluid  becoming  uniformly  turbid,  and  a  slight 
film  forms  on  the  surface,  and  these  cultures  give  a  marked  indole 
reaction  on  the  addition  of  sulphuric  acid  alone,  in  this  respect 
resembling  the  Koch  vibrio.  The  S.  Metchnikovi  is  pathogenic 
to  chickens,  pigeons  and  guinea-pigs,  but  not  to  rabbits  or  mice 
except  in  large  doses.  It  is,  however,  more  pathogenic  to  guinea- 
pigs  than  the  cholera  vibrio.  Pigeons  are  killed  by  intra -muscular 
inoculation,  and  fowls  are  susceptible  to  feeding,  whereas  the 
cholera  vibrio  is  not  pathogenic  to  fowls  by  feeding.  It  is  not 
agglutinated  with  cholera-immune  serum.  Abbott  *  isolated  a 
pathogenic  spirillum  from  the  Schuylkill  River,  Philadelphia,  which 
resembles  the  S.  Metchnikovi  closely,  and  is  probably  identical  with  it. 


Spirillum  Finkleri  (of  Finkler  and  Prior) 

Isolated  from  the  stools  in  certain  cases  of  cholera  nostras,  but 
its  etiological  significance  is  doubtful.  It  occurs  as  short,  thickish, 
curved  or  straight  rods,  and  sometimes  as  spiral  filaments.  It  is 
aerobic  and  facultatively  anaerobic,  does  not  form  spores,  and  does 
not  stain  by  Gram's  method.  In  a  gelatin  stab-culture  a  yellowish 
growth  forms  with  rapid  liquefaction  (Plate  XVIII.  d).  On  agar  a 
thick,  slightly  brownish,  moist  layer  develops.  Serum  is  rapidly 
liquefied.  On  potato  a  slimy  brownish  growth  occurs  even  at 
room  temperature.  It  grows  in  broth  and  peptone -water,  pro- 
ducing a  general  turbidity.  It  does  not  as  a  rule  give  the  indole 
reaction  with  sulphuric  acid  alone,  but  the  ordinary  laboratory 
cultures  after  three  to  four  days'  growth  occasionally  give  a  slight 
reaction.  It  is  stated  to  be  pathogenic  to  guinea-pigs  by  intra- 
peritoneal  inoculation. 

1  Journ.  of  Exper.  Med.,  vol.  i,  1896,  p.  419. 


SPIRILLUM  TYROGENUM  449 


Spirillum  tyrogenum 

Obtained  by  Deneke  from  old  cheese,  and  frequently  spoken  of 
as  Deneke's  spirillum.  It  forms  curved  rods  and  spiral  filaments 
somewhat  closely  resembling  the  Koch  vibrio.  It  grows  well  on 
the  ordinary  culture  media  at  room  temperature,  but  development 
is  usually  slight  or  absent  at  37°  C.  In  a  gelatin  stab -culture  a 
yellowish  growth  occurs  with  liquefaction,  which  is  much  more 
rapid  than  that  of  the  Koch  vibrio,  but  less  so  than  that  of  the 
Finkler-Prior  spirillum.  On  agar  a  thinnish,  brownish,  somewhat 
membranous  and  coherent  layer  slowly  develops  at  room  tempera- 
ture. On  potato  a  yellowish  growth  occurs.  It  is  stated  to  be 
slightly  pathogenic  to  guinea-pigs  by  intra-peritoneal  inoculation. 


Spirillum  rubrum 

A  chromogenic  spirillum  obtained  by  Koch  from  the  putrefying 
tissues  of  a  mouse.  In  a  gelatin  stab-culture  a  dark  red  growth 
slowly  develops  along  the  line  of  puncture  without  liquefaction  ; 
at  the  surface,  however,  the  growth  is  colourless.  In  broth  at 
37°  C.  it  grows  freely,  producing  a  general  turbidity  with  a  red 
deposit  at  the  bottom  of  the  tube  ;  there  is  no  film  formation.  In 
such  a  broth  culture  large  numbers  of  typical  spirillar  filaments 
can  be  seen,  which  are  thin  and  delicate,  of  varying  length,  and 
actively  motile.  It  is  non- pathogenic. 

Vibrios  are  common  in  the  mouth,  and  may  be  met  with  in  the 
discharge  of  septic  ulcers. 


CHAPTER  XV 

STREPTOTHRIX  INFECTIONS— ACTINOMYCOSIS— MY- 
CETOMA— LEPTOTHRIX  BUCCALIS— CLADOTHRIX  DI- 
CHOTOMA— MYCOSIS  TONSILLARIS 

Streptothrix  Infections  (Streptothricosis)  l 

THE  Streptotrichese  are  a  group  of  thread-forming  organisms  showing 
true,  but  not  dichotomous,  branching.  Their  exact  position  in  the 
botanical  scale  is  uncertain  ;  by  some  they  are  considered  to  belong 
to  the  higher  Schizomycetes,  forming  a  connecting  link  between 
these  and  the  Hyphomycetes  ;  others  place  them  among  the  latter, 
and  others  make  them  a  separate  and  distinct  group. 

The  Streptotrichese  form  a  filamentous  network,  or  mycelium, 
the  individual  threads  of  which  show  branching,  while  their  terminal 
portions  undergo  segmentation,  with  the  formation  of  rounded 
bodies  regarded  as  spores.  The  mycelial  network,  unless  old, 
stains  by  Gram's  method,  and  occasionally  possesses  "  acid-fast  " 
properties.  The  leprosy  bacillus  apparently  sometimes  grows  as  a 
streptothrix,  and  the  tubercle,  glanders,  and  perhaps  diphtheria, 
bacilli  may  belong  to  this  group. 

Pathogenic  streptothrix  forms  are  not  uncommon,  the  best 
known  being  those  causing  actinomycosis  of  the  ox  and  other 
animals  and  of  man,  the  white  variety  of  mycetoma,  the  S.  Eppingeri, 
more  or  less  acid-fast,  originally  isolated  from  a  cerebral  abscess, 
and  also  causing  a  variety  of  madura  foot,  S.  Nocardii  of  the  ox, 
and  S.  canis  of  the  dog.  Doubtless  cases  of  streptothrix  infection 
in  man  may  occasionally  be  missed,  as  the  clinical  characters  closely 
resemble  those  of  tuberculosis. 

Pinoy  2  distinguishes  "Actinomycosis,"  in  which  the  grains  in 
the  pus  are  formed  by  very  thin,  unsegmented  mycelial  filaments, 

1  See  Musgrave,  Clegg  and  Polk,  Philippine  Jomn.  of  Science,  vol.  iii, 
1908,  p.  447  ;   Foulertori,  Lancet,  1910,  vol.  i,  p.  551,  et  seq. 

2  Actinomycosis   and   Mycetoma,  Bull,  de  VInst.  Pasteur,  xi,  1913, 
pp.  929,  977. 

450 


ACTINOMYCOSIS  451 

and  "  Mycetomata,"  in  which  the  grains  are  formed  by  thicker 
mycelial  filaments,  segmented,  and  with  a  well-defined  membrane. 


Actinomycosis 

Actinomycosis  in  man  clinically  and  pathologically 
closely  resembles  tuberculosis,  with  which  in  the  past  it 
was  frequently  confounded. 

Actinomycosis  in  cattle  has  long  been  known,  but  its 
exact  pathology  was  involved  in  considerable  doubt  until 
the  researches  of  Bellinger  in  1876.  It  forms  tumours 
chiefly  affecting  the  tongue,  jaw,  face,  and  throat,  and 
was  described  under  such  varied  names  as  wen,  scrofula, 
scirrhus,  osteo-sarcoma,  cancer,  wooden  tongue,  etc. 

The  tumours  after  a  time  break  down  and  discharge, 
the  tongue  often  protrudes  from  the  mouth,  the  saliva 
drips,  and  the  animal  becomes  much  emaciated. 

On  cutting  into  a  "  wooden  tongue,"  or  wen,  a  grating 
sensation  is  felt,  such  as  that  experienced  in  cutting  a 
turnip  or  unripe  pear  ;  on  examining  the  section  little 
rounded,  yellowish,  frequently  almost  caseating  areas  will 
be  noticed,  resembling  old  tubercles.  On  making  sections 
and  examining  with  a  low  power,  these  rounded  areas  are 
found  to  be  composed  of  masses  of  small  round-cells,  with 
occasionally  giant-cells,  surrounded  by  a  capsule  of  fibrous 
tissue.  The  growth  may  be  so  soft  as  to  be  practically 
purulent,  and  abscesses  varying  in  size  from  a  pin's  head 
to  that  of  an  orange  may  be  present  in  the  affected  areas. 
Like  tubercles,  the  growths  may  become  caseous,  calcified, 
or  fibrous.  In  the  growth  or  in  the  pus  from  abscesses, 
when  examined  fresh  with  a  low  power,  yellowish  or 
yellowish- white  granules  will  be  found  here  and  there, 
which  may  be  very  minute,  or  as  large  as  a  small  pin's  head, 
are  somewhat  soft  in  consistence,  and  on  slight  pressure 
flatten  out.  Examined  with  a  high  power,  these  granules 


452  A  MANUAL  OF  BACTERIOLOGY 

are  found  to  contain  round,  ovoid,  or  reniform  bodies 
which  have  a  rosette-like  appearance,  a  more  or  less 
structureless  centre  with  club-shaped  bodies  radially 
arranged  around  the  periphery  (Plate  XIX.  a).  These 
peculiar  structures  are  the  cause  of  the  disease,  and  are 
the  form  assumed  in  the  animal  body  by  an  organism 
belonging  to  the  streptothrix  group  termed  the  Actino- 
myces,  or  Streptothrix  bovis  (Nocardia  bovis),  or,  from  its 
appearance,  the  ray  fungus. 

Sections  of  the  diseased  tissues  show  the  structure  of 
the  organism  still  better.  Gram's  method  usually  gives 
good  results,  and  it  will  generally  be  found  that  the  fol- 
lowing appearances  can  be  observed  :  Surrounded  by  the 
round- cells  are  the  reniform  or  ovoid  bodies,  situated  at 
the  periphery  of  which  are  radially  arranged,  club-shaped 
structures  deeply  stained  with  the  gentian  violet,  while 
the  central  portion  is  unstained  and  structureless,  or 
contains  granular  matter  or  calcareous  particles.  Various 
appearances  may  be  met  with  in  different  parts  of  the 
section,  according  as  the  actinomycotic  nodules  are  cut 
through  their  centre  or  periphery ;  when  the  latter  is  the 
case,  the  clubs  are  shown  in  transverse  section  and  appear 
as  closely  packed,  deeply  stained  dots.  Sometimes,  how- 
ever, in  addition  to  the  clubs,  the  centre  of  the  rosette  is 
occupied  by  numerous  interlacing  filaments,  also  stained 
by  the  gentian  violet. 

In  man,  actinomycosis  is  usually  associated  with  sup- 
puration. If  a  little  of  the  pus  be  examined  it  will  probably 
contain  tiny  yellowish  or  sulphur- yellow  granules,  which, 
microscopically,  are  found  to  consist  of  tufts  of  fine  tangled 
filaments,  the  ends  of  which  may  be  continued  into  little 
swellings  or  clubs.  In  teased-up  specimens,  or  in  sections 
stained  by  Gram's  method,  an  appearance  is  observed 
very  different  from  that  of  the  bovine  variety,  viz.  tufts  of 
interlacing  filaments  stained  by  the  gentian  violet,  but  a 


PLATE  XIX. 


•  •    ^;'-^--1 

^^"">"^-ik 

M6&ft&&. 


a.  Actinomycosisbovis.     Section  of  tongue.     Gram.      X  350. 


•  *- '  *  *  jt**£ 


6.  Mycetoma.     Section  of  tissue,"  white  variety.     Cram.      X  350. 


ACTINOMYCOSIS  453 

complete  absence  of  purple  clubs  (Plate  XX.  a).  Clubs, 
however,  are  frequently  present  around  the  periphery  of 
the  filamentous  tufts  in  a  stunted  condition,  although  they 
do  not  usually  stain  by  Gram's  method.  These  clubs  are 
often  seen  better  in  fresh  specimens  of  the  pus  or  in 
unstained  sections,  or  by  staining  with  orange-rubin,  or 
the  Ehrlich-Biondi  reagent  (Plate  XX.  6).  The  con- 
ditions in  cattle  and  man,  at  first  sight  so  very  different , 
are  thus  seen  to  be  similar,  a  similarity  which  is  further 
established  by  the  occasional  occurrence  in  cattle  of 
filamentous  tufts,  staining  by  Gram's  method,  within  the 
rosettes,  and  by  the  clubs  in  man  now  and  then  taking 
on  the  gentian- violet  stain. 

Cultural  characters. — The  cultivation  of  the  Actino- 
myces  can  be  performed  by  collecting  the  pus  from  a  case 
of  the  disease  in  sterilised  tubes,  and  subsequently  turning 
it  out  into  a  sterilised  capsule  and  picking  out  the  actino- 
mycotic  granules  with  sterilised  needles,  planting  these 
on  the  surface  of  glycerin  agar,  and  incubating  at  37°  C. 
A  certain  number  of  the  tubes  will  probably  be  uncon- 
taminated,  but  in  others  a  growth  of  the  Micrococcus 
pyogenes  var.  aureus  or  other  pyogenic  organism,  which  is 
not  unfrequently  associated  with  the  Actinomyces,  may 
occur.  In  the  uncontaminated  tubes  a  growth  begins  to 
appear  in  a  few  days  in  the  form  of  little  colonies  of  a  tough 
membranous  consistence,  somewhat  wrinkled,  greyish,  and 
shining,  while  the  agar  beneath  them  becomes  stained 
brownish.  The  growth  increases  and  the  colonies  coalesce, 
forming  a  brownish,  wrinkled,  membranous  expansion, 
sticking  firmly  to  the  agar^and  difficult  to  remove  or 
break  up,  while  the  agar  becomes  stained  brown  through- 
out ;  later  on  the  membranous  growth  may  become 
dappled  with  yellow  as  though  powdered  with  flowers  of 
sulphur,  but  occasionally  remains  whitish.  In  gelatin 
little  spherical  feathery  tufts  develop,  and  sink  to  the 


454  A  MANUAL  OF  BACTERIOLOGY 

bottom  as  liquefaction  progresses.  On  potato  a  remark- 
able growth  develops  ;  at  first  brownish,  it  afterwards 
becomes  almost  black,  and  is  very  thick  or  heaped  up  with 
a  much  wrinkled  surface,  while  later  on  it  has  the  appear- 
ance of  being  sprinkled  with  flowers  of  sulphur  (Fig.  49). 
In  broth  delicate  woolly  flocculi  form. 
Films  from  young  agar  cultures  show 
masses  of  tangled  filaments,  which  appear 
to  be  more  or  less  branched,  and  stain 
well  with  the  ordinary  anilin  dyes  and  by 
Gram's  method  ;  with  the  latter  the  fila- 
ments often  appear  somewhat  beaded, 
but  no  trace  of  rosette  formation  or  even 
of  clubs  is  ever  found  in  cultures  (Fig.  50). 
In  pus,  especially  human,  the  filaments 
can  sometimes  be  seen  if  stained  by 
Gram's  method  with  orange-rubin.  Inocu- 
lated into  the  peritoneal  cavity  of  rabbits 
"  ^  and  guinea-pigs  the  cultivated  organism 
reproduces  the  disease,  numerous  actino- 
mycotic  nodules  forming  in  the  peritoneum 
and  elsewhere.  There  is  much  doubt  as 
to  the  mode  of  spread  of,  and  the  infection 
FIG.  49.— Actino-  of  man  with,  the  disease.  It  does  not  seem 
myoes.  Potato  to  be  particularly  contagious,  and  diseased 
months  old.  *  anc^  healthy  animals  are  often  placed  to- 
gether without  bad  result ;  it  can,  however, 
be  conveyed  by  direct  inoculation,  for  calves  inoculated 
intraperitoneally  with  portions  of  diseased  tissues  die  after 
some  weeks  or  months,  with  an  abundant  development 
of  actinomycotic  nodules,  as  shown  by  the  experiments 
of  Jone  and  Ponfick.  Crookshank  also  infected  a  calf  with 
the  material  from  a  human  case.  Feeding  experiments 
give  negative  results.  The  view  generally  held  is  that 
the  organism  occurs  on  cereals,  straw,  or  roots,  and  gains 


PLATE  XX. 


a.  Actinomycosis  hominis. 
mycelial  mass. 


Section  of  liver  showing 
Gram,      x  500. 


b. 


clubs.     Gram,      x  350. 


Section  showing  a  ring  of  stunted 
Same  material  as  Fig.  a  above. 


THE  ACTINOMYCES 


455 


access  to  the  system  through  slight  scratches  or  wounds 
in  the  mucous  membrane  of  the  mouth,  pharynx,  or  larynx. 
In  man  no  source  of  infection  has  been  traced,  though 
cases  have  been  reported  where  the  disease  has  occurred 
after  eating  grains  of  barley,  etc.  The  disease  is  met  with 
not  only  in  cattle,  but  also  in  horses  and  swine.  In  the 
last-named  animals  considerable  calcification  may  be 


FIG.  50. — Actinomyces.     Film  preparation. 
Gram.      X  750. 


present  in  the  nodules,  and  it  may  be  necessary  to  decalcify 
with  dilute  nitric  or  hydrochloric  acid  before  the  rosettes 
can  be  stained. 

It  is  important  to  note  that  tuberculin  may  cause  a 
reaction  in  actinomycosis,  similar  to  that  which  occurs 
in  tuberculosis,  and  as  the  actinomycotic  lesions  are  very 
like  those  which  are  found  in  the  latter  disease,  mistakes 
may  easily  be  made,  and  can  only  be  avoided  by  a  micro- 
scopical examination.  It  is  of  considerable  practical 


456  A  MANUAL  OF  BACTERIOLOGY 

importance  to  distinguish  actinomycosis  from  tuberculosis, 
for  in  many  cases  of  the  former,  both  in  man  and  in  animals, 
iodide  of  potassium  exerts  a  specific  curative  action. 
Vaccine  treatment  has  also  been  employed  with  a  certain 
amount  of  success. 

By  some  several  species  of  Actinomyces  are  believed  to  exist, 
but  Homer  Wright x  considers  that  but  one  species  of  micro- 
organism is  the  etiological  agent,  both  in  man  and  animals,  the 
A.  bovis.  Pinoy  regards  Actinomycosis  in  man  as  caused  by  several 
fungi  (Nocardia,  Indiella,  Colmistreptothrix). 

"  Farcin  des  bceufs,"  a  disease  of  cattle  occurring  in  Guadeloupe, 
and  characterised  by  infection  first  of  the  skin  and  afterwards  of 
the  lymphatic  glands  and  viscera,  is  due  to  the  S.  Nocardii. 


Clinical  Examination 

1.  Pour  out  the  pus  or  discharge  into  a  large  capsule  or  Petri 
dish  so  that  it  forms  a  thin  layer,  look  for  any  yellowish  or  other 
granules,  pick  them  out  with  a  needle,  and  place  on  a  clean  slide 
in  a  drop  of  50  per  cent,  glycerin.  If  no  granules  can  be  found,  a 
little  of  the  discharge  may  be  spread  on  a  slide  with  a  drop  of  50  per 
cent,  glycerin.  Cover  with  a  cover-glass,  and  apply  a  little  pressure. 
Examine  with  a  f-in.  objective.  If  any  actinomycotic  tufts  are 
present  they  will  be  seen  as  yellowish  or  pale  brownish,  spheroidal, 
ovoid,  or  reniform  masses,  and  with  a  ^-in.  objective  will  be  found 
to  have  a  radiating  structure  from  the  presence  of  the  clubs. 

2.  Stain  films  of  the  discharge,  by  Gram's  method,  with  eosin. 
The  actinomycotic  tufts  will  generally  be  found  to  consist  of  little 
masses  of  tangled  filaments  stained  violet,  and  surrounded  by  a 
pink  zone  which  has  an  indistinct  radiating  structure. 

N.B. — In  most  instances  the  clubs  in  Actinomycosis  hominis  do 
not  stain  by  Gram's  method.  The  reverse  is  the  case  in  Actino- 
mycosis bovis. 

3.  Sections  of  actinomycotic  tissue  are  best  prepared  by  the 
paraffin  method.     If  frozen,  the  actinomycotic  nodules  are  very 
apt  to  fall  out.     Sections  may  be  stained  by  any  of  the  following 
ways  : 

(a)  By  Gram's  method,  with  eosin  or  orange-rubin. 

(6)  With  the  Ehrlich-Biondi  triple  stain.    Stain  for  from  half  an 

1  Journ.  Med.  Research.  1905. 


MADUKA  DISEASE  457 

hour  to  two  hours.  Place  in  methylated  spirit  until  the  sections 
appear  greenish,  then  pass  through  absolute  alcohol  and  xylol. 
The  clubs  are  stained  yellowish-brown,  and  are  sometimes  shown 
in  human  cases  when  unstained  by  Gram's  method. 

(c)  By  Plant's  method.     Stain  in  warm  carbol-fuchsin  for  ten 
minutes,  rinse  well  in  water,  stain  in  a  saturated  solution  of  picric 
acid  in  methylated  spirit  for  five  to  ten  minutes,  rinse  well  in  water, 
place  in  50  per  cent,  alcohol  for  ten  minutes,  pass  through  absolute 
alcohol  and  xylol. 

(d)  Good  preparations  may  be  obtained  by  staining  in  Ehrlich's 
haematoxylin  and  counter-staining  with  orange  rubin.     This  may 
also  show  the  clubs  when  they  are  unstained  by  Gram's  method. 


Madura  Disease  or  Mycetoma 

Madura  disease,  otherwise  known  as  madura  foot,  mycetoma,  or 
the  "fungus  disease  of  India,"  is  a  chronic  local  affection  generally 
attacking  the  foot,  occasionally  the  hand,  sometimes  extending  up 
the  leg,  but  rarely  to  the  trunk.  The  disease  occurs  in  certain 
districts  in  India,  and  full  descriptions  of  it  have  been  given  by 
Vandyke  Carter  and  by  Lewis  and  Cunningham.  A  "  madura  " 
foot  appears  enlarged,  and  numerous  sinuses  with  raised  mammilated 
apertures  open  on  the  surface  (Fig.  51).  On  making  a  section  into 
the  diseased  tissues  the  bones  are  found  to  be  more  or  less  carious, 
while  the  soft  structures  are  tough  and  hypertrophied  from  the 
occurrence  of  chronic  inflammatory  changes.  Numerous  small 
cavities  are  present,  sometimes  filled  by  yellowish  granules  resem- 
bling fish-roe,  and  hence  termed  "  roe -like  particles,"  at  others 
containing  black  particles  of  irregular  shape,  coal -like  consistence, 
and  variable  size,  exceptionally  as  large  as  a  marble  or  walnut. 
The  presence  of  the  white  or  black  granules,  which  may  be  dis- 
charged from  the  sinuses  before  mentioned,  divides  the  disease  into 
two  classes — the  so-called  white  and  black  varieties.  Lewis  and 
Cunningham  have  also  described  a  third  variety,  in  which  the 
granules  are  red  like  cayenne  pepper. 

Vandyke  Carter  l  first  called  attention  to  the  similarity  between 
the  white  variety  and  actinomycosis  in  their  microscopical  characters. 
In  sections  stained  by  Gram's  method  more  or  less  crescentic  or 
reniform  bodies  are  noticeable,  divided  into  wedge-shaped  areas, 
which  contain  masses  of  fine  filaments  stained  purple.  Surrounding 

1  Bombay  Med.  and  Phys.  Soc.,  vol.  ix,  1886  (new  series),  p.  86.  Also 
Hewlett,  Trans.  Path.  Soc.  Lond.,  vol.  xlii,  1893. 


458  A  MANUAL  OF  BACTERIOLOGY 

the  crescentic  bodies  is  a  zone  of  radially  arranged  elements,  many 
of  which  are  fan-shaped  owing  to  branching  ;  they  are  indistinct, 
as  they  do  not  stain  with  the  gentian  violet,  but  they  are  very 
suggestive  of  the  club-shaped  structures  present  in  actinomycosis, 
and  they  resemble  the  Actinomycosis  hominis  inasmuch  as  they  do 
not  stain  by  Gram's  method  (Plate  XVIII.  6).  By  staining  with 
haematoxylin  and  orange  rubin,  or  with  the  Ehrlich-Biondi  triple 
stain,  here  and  there  in  the  radial  zone  well-defined  clubs  can  be 
demonstrated.  It  seems,  therefore,  that  the  radial  zone  is  composed 
of  degenerate  club-shaped  structures,  and  the  disease  evidently 


FIG.  51. — A  foot  affected  with  madura  disease.     (White  variety.) 

closely  resembles  actinomycosis,  but  seems  to  be  due  to  a  different 
species  of  streptothrix. 

From  a  case  of  the  white  variety  1  Boyce  cultivated  a  streptothrix 
which  differed  somewhat  from  the  Actinomyces,  as  it  grew  slower, 
produced  no  pigment,  and  on  agar  formed  white  raised  colonies 
with  radial  grooves,  not  unlike  the  tiny  barnacles  found  on  wooden 
piles  in  the  sea.  Vincent 2  also  isolated  a  streptothrix,  perhaps 
identical  with  that  of  Boyce,  which  differed  from  the  Actinomyces 
in  growing  feebly  in  broth,  in  not  liquefying  gelatin,  and  in  not 
being  inoculable  in  the  rabbit.  He  describes  it  as  forming  on 
glycerin  agar  umbilicated  colonies,  first  white  and  afterwards  red. 
Shattock  3  suggests  that  the  red,  cayenne -pepper-like  grains  occa- 
sionally met  with  in  mycetoma  may  be  due  to  colonies  of  the  strepto- 

1  Hygienische  Rundschau,  1894,  No.  12. 

2  Ann.  de  VInst.  Pasteur,  1893. 

3  Trans.  Path.  Soc.  Lond.,  vol.  xlix,  1898,  p.  294. 


MADURELLA  459 

thrix  which  have  produced  their  pigment.  Microscopically,  this 
organism  (Streptothrix  madurce,  Nocardia  madurce)  is  identical  with 
the  Actinomyces.  Musgrave  and  Clegg  in  a  case  of  the  white 
variety  isolated  a  streptothrix  (S.  freeri)  differing  from  the  S. 
madurce,  but  identical  with  the  S.  Eppingeri  (Nocardia  asteroides). 

The  relation  of  the  black  to  the  white  variety  of  madura  disease 
has  been  somewhat  debated.  Kanthack x  described  the  black 
variety  as  being  probably  a  late  stage  of  the  white.  It  seems, 
however,  that  the  co -existence  of  the  two  conditions  in  the  same 
specimen  is  very  rare,  and  Boyce  and  Surveyor,2  after  a  critical 
examination  of  a  large  number  of  specimens,  came  to  the  conclusion 
that  the  black  variety  is  a  distinct  disease,  and  due  to  an  organism 
belonging  to  the  group  of  the  higher  fungi,  the  black  particles  or 
masses  being  the  lignified  mycelium  or  "  sclerotium  "  such  as  is 
met  with  in  ergot. 

Pinoy  regards  the  white  variety  as  an  Actinomycosis,  the  black 
variety  as  a  Mycetoma. 

It  is  difficult  experimentally  to  reproduce  mycetoma  in  animals, 
but  Pinoy  has  succeeded  in  doing  so  with  an  Aspergillus,  and 
Xicolle  with  Madurella  tozeuri  (North  Africa),  both  in  pigeons. 

By  planting  out  the  granules  from  an  early  case  of  the  black 
variety  Wright  succeeded  in  cultivating  a  hyphomycete. 3  It 
formed  long  branching  hyphse,  but  no  spore-bearing  organs  were 
produced,  and  inoculation  experiments  on  animals  were  negative. 
It  grew  on  potato  as  a  dense,  widely  spreading,  coherent,  velvety 
membrane,  in  colour  pale  brown  with  white  periphery.  Small 
drops  of  brown,  coffee-coloured  fluid  appeared  on  the  surface,  and 
the  potato  became  brown  throughout.  On  agar  the  growth  formed 
a  meshwork  of  widely  spreading  greyish  filaments  ;  in  old  cultures 
(also  in  potato  infusion)  black  hard  granules,  or  "  sclerotia,"  were 
observed.  In  broth  little  balls  of  radiating  filaments  developed. 

It  would  seem  that  there  are  several  conditions,  both  in  actino- 
mycosis  and  in  mycetoma,  having  a  general  resemblance  but 
differing  slightly,  and  dependent  upon  different  species  of  parasitic 
organism. 

According  to  Pinoy  (loc.  cit.},  the  Mycetomata  are  caused  by  fungi 
belonging  to  the  genera  Madurella,  Aspergillus,  and  Sterigmato- 
cystis.  The  common  form  in  the  Indian  and  African  Mycetoma  is 
Madurella  mycetomi  (Laveran). 

1  Journ.  Path,  and  Bact.,  1892. 

2  Proc.  Roy.  Soc.  Lond..  1893,  and  Phil.  Trans.  Roy.  Soc.  Land. 

3  Journ.  Exp.  Med.,  vol.  iii,  1898,  p.  421. 


460  A  MANUAL  OF  BACTERIOLOGY 

Mycosis  tonsillaris  (Mycosis  pharyngis  lepto- 
thricia) 

A  chronic  disease  attacking  young  adults,  resistant  to  treatment, 
and  characterised  by  the  presence  of  small,  white,  tough,  adherent 
excrescences  on  the  mucous  membrane  of  the  pharynx.  Micro- 
scopically, the  patches  consist  of  collections  of  epithelial  cells  and 
debris,  infiltrated  with  leptothrix  filaments  and  bacteria.  The 
disease,  however,  seems  to  be  a  keratosis,  infection  with  the 
organisms  being  secondary. 

But  occasionally  a  true  "  mycosis  "  apparently  occurs,  readily 
amenable  to  treatment,  and  due  to  a  leptothrix.1 

Leptothrix  buccalis 

Four  somewhat  similar  thread  forms  occur  in  the  mouth,  viz. 
Leptothrix  racemosa,  L.  buccalis  maxima,  L.  innominata,  and 
Bacillus  maximus  buccalis.  The  first  is  very  common,  forms  large 
threads,  shows  a  peculiar  beaded  appearance  on  staining  which  has 
been  regarded  as  sporulation,  and  may  be  a  fungus  form.  L.  buccalis 
maxima  and  L.  innominata  differ  from  each  other  in  that  the  former 
gives  a  blue  granulose  reaction  when  treated  with  iodine  and  dilute 
sulphuric  acid,  while  the  latter  does  not.  All  these  three  organisms 
are  very  similar,  and  the  filaments  are  either  unsegmented,  or  the 
segments  are  of  considerable  length.  The  B.  maximus  buccalis  is 
very  like  the  L.  buccalis  maxima,  but  does  not  give  the  granulose 
reaction,  and  its  segments  are  shorter.  It  is  motile,  flagellated,  and 
sporing,  and  stains  by  Gram's  method. 

Some  confusion  exists  respecting  the  thread  forms  of  the  mouth.2 

Cladothrix  dichotoma 

An  organism  not  unfrequently  met  with  in  natural  waters.  It 
forms  long  threads,  straight,  or  sometimes  slightly  undulating,  or 
even  spiral  and  apparently  branched,  though  the  branching  is  not 
dichotomous.  It  can  be  cultivated  on  the  ordinary  laboratory 
media  at  room  temperature,  forming  on  agar  a  brownish,  wrinkled, 
tough,  membranous  layer,  very  adherent,  and  staining  the  medium 
beneath  it  a  pale  brown,  not  unlike  the  Actinomyces  in  these  respects. 
It  is  non-pathogenic. 

1  See  Glasgow  Medical  Journal,  No.  2,  1896,  p.  81  et  seg.  (Brown 
Kelly).  2  See  Goadby,  Mycology  of  the  Mouth. 


CHAPTER  XVI 

THE  SACCHAROMYCETACE.E 

The  Pathogenic  Blastomycetes — Yeasts  and  Fermentation 

The  Yeasts 

THE  Saccharomycetacese  or  Yeasts  are  characterised  by  a  vegeta- 
tive reproduction  by  budding  or  gemmation.  If  a  cell  of  ordinary 
brewer's  yeast  be  watched  under  conditions  favourable  to  growth 
and  reproduction,  it  will  be  found  that  a  slight  protuberance  makes 
its  appearance  at  one  pole  of  the  organism  ;  this  increases  in  size, 
and  ultimately  a  daughter-cell  resembling  the  parent  is  reproduced 
and  separates  off. 

The  true  yeasts  also  reproduce  by  spore -formation  by  ascospores 
(p.  465)  ;  in  some  there  is  a  fusion  of  cells  before  sporulation,  in 
others  the  first  cell  formed  by  germination  of  the  spore  undergoes 
fission,  forming  what  is  known  as  a  pro-mycelium,  after  which  the 
cells  multiply  by  gemmation.  The  Saccharomycetaceae  may  there- 
fore be  divided  into : 

1.  Zygosaccharomyces,  in  which  pairs  of  cells  fuse  before  sporula- 
tion. 

2.  Saccharomyces,  in  which  there  is  no  fusion  of  cells  before 
sporulation,  and  in  which  the  spores  germinate  by  ordinary  budding. 

3.  Saccharomycoides,  in  which  the  spores  germinate  by  means  of 
a  promycelium. 

Besides  the  true  yeasts,  there  are  a  number  of  budding  forms 
known  which  do  not  spore.  These  have  been  termed  "  Torulse  " 
(any  yeast-like  cell  is  frequently  called  a  "  torula  ").  Some  form 
films  on  saccharine  li  quids  and  are  known  as  Mycoderma.  Organisms 
are  also  known  having  a  yeast-like  form  and  multiple  spores  but 
multiplying  by  fission  ;  these  have  been  termed  Schizosaccharo- 
myces.  The  position  of  these  forms  is  uncertain  and  they  are 
classed  by  the  botanist  among  the  Fungi  Imperfecti  (p.  470). 

In  addition  to  reproduction  by  gemmation,  the  Saccharomyce- 

461 


462  A  MANUAL  OF  BACTERIOLOGY 

tacese  are  also  distinguished  from  the  Bacteria  by  their  larger  size, 
and  in  those  forms  in  which  endospores  occur  by  the  spores  being 
multiple  and  not  single  in  each  cell  and  by  having  a  cellulose  cell- 
wall.  From  the  Hyphomycetes,  or  moulds,  the  Saccharomycetaceae 
are  distinguished  by  being  unicellular,  and  by  the  reproduction 
being  generally  asexual.  The  Saccharomycetacese,  however,  are 
probably  much  more  nearly  allied  to  the  Hyphomycetes  than  are 
the  Bacteria,  for  many  of  the  moulds  have  a  stage  in  which  the 
mycelium  (see  next  chapter)  resembles  an  aggregation  of  yeast-cells, 
and  the  yeasts  in  old  cultures  form  films  in  which  the  cells  become 
much  elongated,  like  those  in  the  mycelium  of  a  mould.  Jorgensen 
and  others  have  attempted  to  show  that  some  of  the  yeasts  are 
stages  in  the  development  of  a  fungus,  but  it  cannot  be  said  that 
this  has  yet  been  satisfactorily  demonstrated. 


Pathogenic  Yeasts  l 

Organisms  apparently  belonging  to  the  Saccharomyce- 
tacese  and  termed  Blastomycetes  have  been  isolated  from 
certain  tumours,  and  have  been  regarded  as  having  an 
etiological  significance  in  connection  with  malignant 
disease.  Sanfelice  cultivated  yeast  forms  from  fermenting 
fruits,  which,  on  inoculation  into  guinea-pigs,  produced 
death  in  about  a  month  with  the  formation  of  a  tumour 
at  the  seat  of  inoculation  and  embolic  growths  in  the 
spleen  and  liver.  He  also  obtained  a  similar  yeast  from 
an  ox  affected  with  carcinoma,  which  on  subcutaneous 
inoculation  killed  guinea-pigs  in  about  two  months,  and 
inoculated  into  the  peritoneum  in  a  month,  with  multiple 
embolic  growths  in  the  lungs,  spleen,  and  mesenteric  glands. 
A  good  deal  of  calcification  was  present  in  the  growths, 
from  which  fact  Sanfelice  named  this  yeast  Saccharomyces 
litogenes.  Rabinowitch  and  also  Foulerton  2  have  found 
that  some  of  the  ordinary  yeasts  give  rise  to  tumour 
formation  on  inoculation,  especially  in  the  rabbit.  These 

1  See  Le  Count  and  Myers,  Journ.  of  Infectious  Diseases,  vol.  iv, 
1907,  p.  187. 

2  Journ.  Path,  and  Bact.,  vol.  vi,  1899,  p.  37. 


PATHOGENIC  YEASTS  463 

tumours  produced  by  yeasts  are  probably  granulomata  and 
not  true  malignant  tumours. 

Curtis  l  obtained  a  yeast  from  an  apparently  myxo- 
matous  tumour  in  a  young  man.  The  organism  was  met 
with  in  two  forms — free  and  encapsuled.  The  free  form 
appeared  in  young  agar  cultures  as  round  or  ovoid  cells 
measuring  3  to  6  /x  in  diameter,  often  showing  budding. 
The  encapsuled  form  was  met  with  in  the  original  tumour 
and  in  the  tissues  of  inoculated  animals,  and  occurred 
as  a  large  sphere  16  to  20  /u.  in  diameter,  enclosing  the 
yeast  cell,  the  capsule  being  hyaline  and  4  to  6  M  in  thick- 
ness. On  agar  at  37°  C.  the  organism  formed  whitish, 
opaque,  creamy  colonies  in  two  to  three  days,  becoming 
a  thick  creamy  growth  at  the  end  of  a  week,  on  gelatin 
white  colonies  or  growth  in  four  to  five  days  without 
liquefaction,  and  in  broth  a  flocculent  deposit,  the  broth 
remaining  clear.  It  was  aerobic,  did  not  grow  on  serum, 
and  formed  a  small  quantity  of  acetic  acid  and  alcohol 
when  grown  in  beerwort  and  sugar  solutions.  It  was  not 
pathogenic  for  guinea-pigs,  but  inoculated  into  rabbits, 
rats,  mice,  and  dogs  it  produced  tumours  and  caused  death. 
The  tumours  to  the  naked  eye  appeared  to  be  myxo- 
sarcomata,  and  in  them  the  yeasts  were  found. 

Busse  also  obtained  a  pathogenic  yeast  from  a  young 
woman  who  suffered  from  a  tumour  of  the  tibia,  and 
ultimately  died  with  diffused  growths  in  the  bones  and 
organs.  The  yeast-like  cells  were  observed  in  the  affected 
parts,  and  were  isolated  by  cultivation,  and  the  cultures, 
inoculated  into  mice  and  rabbits,  produced  death  with 
growths  in  the  organs.  As  in  Curtis's  case,  the  cells  in 
the  tissues  appeared  to  be  encapsuled. 

Gilchrist  described  a  case  of  blastomycetic  dermatitis. 
Small  miliary  abscesses  were  present  in  the  rete  and 
corium,  in  the  pus  of  which  the  parasitic  cells  were 
1  Ann.  de  Vlnst.  Pasteur,  x,  1896,  p.  449  (Refs.). 


464  A  MANUAL  OF  BACTERIOLOGY 

observed.  These  were  usually  in  pairs  of  unequal  size, 
the  largest  measuring  about  16  /*,  surrounded  by  a  well- 
defined  capsule,  and  containing  a  granular  protoplasm  in 
which  a  vacuole  was  present.  Clinically,  the  case  had 
been  regarded  as  one  of  scrofuloderma,  but  no  tubercle 
bacilli  could  be  found. 

Numerous  cases  of  blastomycetic  dermatitis  have  now 
been  recognised,  and  several  instances  of  general  systemic 
blastomycetic  infection  have  been  recorded. 

Granulomatous  tumours  occurring  in  epidemics  among 
horses  in  Japan,  France,  and  Italy  are  also  caused  by 
Blastomycetes. 

Clinical   Examination   (Pathogenic  Yeasts,    etc.) 

The  cells  can  be  well  seen  in  the  fresh  state  in  the  teased-up 
tissues  mounted  in  water  or  glycerin. 

Curtis  recommends  staining  in  carbol-thionine  blue,  and  for 
sections,  picro- carmine. 

Busse's  method  for  sections  is  as  follows  : 

1.  Haematoxylin  solution  for  fifteen  minutes. 

2.  Wash  in  distilled  water. 

3.  Counter-stain  in  weak  carbol-fuchsin  (1  :  20)  for  thirty  minutes 
to  twenty -four  hours. 

4.  Decolorise  in  95  per  cent,  alcohol  for  fifteen  seconds  to  one 
minute. 

5.  Absolute  alcohol,  xylol,  mount  in  Canada  balsam. 
Gilchrist  recommends  treating  the  sections  with  10  per  cent. 

caustic  potash  solution  and  examining  in  50  per  cent,  glycerin 
without  staining. 

Brayton  recommends  that  small  pieces  of  the  tissues  should  be 
excised  from  the  growing  margin,  treated  with  ether  for  two  to 
five  minutes,  macerated  in  20  to  30  per  cent,  caustic  potash  solution 
for  five  to  ten  minutes,  and  then  examined  without  staining. 
Cultures  may  be  readily  obtained,  with  a  little  care,  preferably  on 
beer-wort  gelatin  or  maltose  agar. 


FERMENTATION  465 


Fermentation 

The  yeasts  are  of  great  importance  in  inducing  many  chemical 
changes,  especially  alcoholic  fermentation,  beer  and  wine  being 
almost  exclusively  due  to  their  activity. 

Taking  brewer's  yeast,  Saccharomyces  cerevisice,  as  a  type,  the 
yeast  cell  is  observed  to  be  slightly  ovoid  in  shape,  measuring  8  to 
9  p.  in  diameter.  The  protoplasm  is  granular,  contains  one  or  more 
clear  spaces  or  vacuoles,  frequently  bright,  refractile  globules  of 
fatty  matter,  and  is  surrounded  by  a  cell  wall  of  cellulose.  It  has 
been  repeatedly  stated  that  a  nucleus  is  present,  but  this  is  doubtful. 
When  the  yeast-cell  is  freely  supplied  with  nutriment,  reproduction 
by  gemmation  proceeds  rapidly,  and  a  whole  string  of  cells  may 
form  owing  to  the  daughter-cells  budding  again  before  they  have 
separated  from  the  parent.  When  the  cell  is  starved,  gemmation 
ceases,  fat-globules  and  vacuoles  increase  in  number,  and  the  cell 
may  finally  become  little  more  than  a  large  vacuole,  the  protoplasm 
forming  a  thin  coating  over  the  inside  of  the  cell  wall.  Within  the 
vacuoles  are  often  seen  minute  spherical  bodies  of  a  doubtful  nature 
in  rapid  movement.  In  ordinary  circumstances  endospore  forma- 
tion does  not  occur,  but  by  deprivation  of  nutriment,  as  by  growing 
on  a  block  of  plaster-of -Paris,  the  cells  develop  spores.  First  the 
cell  becomes  divided  by  the  development  of  membranes,  the  so- 
called  "  partition- wall  formation,"  into  several  chambers  in  which 
the  spores  form.  In  the  different  yeasts  the  number  and  arrange- 
ment of  the  spores  vary  ;  in  the  S.  cerevisice  the  typical  number  is 
four,  arranged  close  together,  three  on  one  plane  and  one  resting 
on  these,  like  a  pyramid  of  billiard  balls. 

Although  the  reproduction  of  yeasts  by  gemmation  or  ascospore 
formation  is  usually  asexual,  ascospore  formation  is  sometimes 
preceded  by  conjugation  of  sister-cells,  or  conjugation  may  occur 
between  neighbouring  cells  at  the  moment  of  germination  (Guillier- 
mond,  Nadson,  and  Marchand). 

The  spores  are  of  considerable  importance  in  the  identification 
of  species  of  Saccharomyces,  as  the  form  of  the  cells  alone  and  the 
growths  on  culture  media  are  not  sufficiently  distinctive.  In  fact 
so  little  can  these  two  characters  be  relied  upon  that  in  order  to 
isolate  in  pure  cultivation  it  is  necessary  to  grow  from  a  single  cell. 
This  can  be  done  by  making  a  miniature  plate  cultivation  with 
wort-gelatin  on  a  large  sterilised  cover-glass,  and,  after  the  layer  of 
gelatin  has  set,  mounting,  gelatin  downwards,  on  a  large  cell  on  a 
glass  slide.  The  cover-glass  should  be  divided  into  small  squares 

30 


466  A  MANUAL  OF  BACTERIOLOGY 

by  cross-lines  etched  on  the  glass  and  numbered.  The  preparation 
is  carefully  examined  with  a  J  or  J  inch  objective,  and  the  positions 
of  single  isolated  cells  are  noted.  This  is  not  a  difficult  matter  on 
account  of  the  comparatively  large  size  of  the  yeast-cells,  and  their 
position  is  determined  by  the  numbered  squares  on  the  cover-glass. 
The  preparations  are  kept  in  a  moist  chamber  in  a  warm  place, 
and  when  visible  colonies  have  developed,  those  which  are  derived 
from  a  single  cell  can  be  inoculated  into  tubes  or  flasks  of  a  suitable 
culture  medium. 

It  is  found  that  the  various  yeasts  form  spores  in  different  periods 
of  time  when  grown  under  similar  conditions,  and  on  this  fact  is 
based  what  is  known  as  the  analysis  of  yeast — a  most  valuable 
method,  which  we  owe  to  Hansen.  The  chief  "  diseases  "  of  beers 
and  yeast — i.e.  abnormal  fermentations  giving  rise  to  inferior  pro- 
ducts— are  due  to  admixture  of  certain  "  wild  yeasts,"  as  they  are 
termed,  with  the  brewer's  yeast,  chiefly  the  S.  ellipsoideus  and 
S.  pastor ianus  ;  and,  in  order  to  detect  these  "  disease  "  species, 
the  analysis  consists  in  determining  at  what  time  ascospores  appear. 
The  mode  of  procedure  is  as  follows  : 

The  yeast  is  sown  in  a  flask  of  sterile  wort,  and  incubated  at 
25°  C.  for  twenty -four  hours.  The  yeast  revives,  and  from  the 
deposit  of  young  cells  two  cultures  are  made  on  plaster-of-Paris 
blocks.  These  cultures  are  kept,  one  at  25°  C.,  the  other  at  15°  C., 
and  are  examined  twice  daily.  In  an  uncontaminated  brewing 
yeast  ascospores  should  not  be  detected  in  less  than  thirty  hours 
in  the  culture  kept  at  25°  C.,  and  seventy-two  hours  in  that  kept  at 
15°  C.  The  plaster-of-Paris  blocks  are  sterilised  by  careful  flaming 
in  the  Bunsen,  and  are  then  placed  in  sterile  glass  capsules  with 
lids,  containing  sufficient  sterilised  water  thoroughly  to  moisten  the 
whole  of  the  blocks  ;  unless  this  is  done  no  growth  occurs.  By 
this  method  of  analysis  as  little  "  wild  yeast  "  as  one  two-hundredth 
of  the  whole  can  be  detected. 

Besides  the  distinct  species  of  yeasts,  there  are  also  a  number  of 
varieties  employed  in  brewing,  etc.,  differing  but  slightly  in 
morphological  and  cultural  characters,  yet  giving  rise  to  varied 
products.  These  varieties  may  be  divided  into  two  groups — the 
surface,  high  or  top,  and  the  sedimentary,  low  or  bottom,  fermenta- 
tion forms.  In  this  country  beer  is  brewed  by  fermenting  an 
infusion  of  malt  ("  wort  ")  with  yeast,  which,  during  fermentation, 
rises  to  the  surface,  and  belongs  to  the  first  group  ;  while  the  German 
beers  are  obtained  by  yeast,  which  sinks  to  the  'bottom,  and  belongs 
to  the  second  group.  The  floating  of  the  yeast  in  the  high  fermenta- 
tion process  seems  to  be  due  to  the  attachment  of  minute  bubbles  of 


FERMENTATION  467 

carbonic  acid  gas  to  the  cells,  and  it  has  not  yet  been  possible  to 
convert  the  one  form  into  the  other. 

Characters  of  some  of  the  more  important  yeasts. — Hansen  divides 
the  important  yeasts  into  groups  having  the  same  general  characters, 
and  distinguishes  the  varieties  in  each  by  Roman  numerals  (I, 
II,  etc.). 

CEREVTSLS:  GROUP. — These  are  the  yeasts  producing  the  normal 
fermentations  resulting  in  beer,  etc.  They  are  round  or  slightly 
ovoid  cells,  and  four  ascospores  are  produced.  In  old  cultures  long 
sausage-shaped  or  even  filamentous  cells  may  be  met  with. 

8.  cerevisice  I.  and  II. — These  are  bottom  fermentation  forms  in 
use  at  the  Old  Carlsberg  Brewery  ;  the  cells  of  No.  II  are  rounder 
and  slightly  larger  than  those  of  No.  I,  and  ascospore  formation  is 
more  abundant. 

There  is  also  a  top  fermentation  form  described  by  Hansen 
(S.  cerevisice  I  top),  which  is  the  yeast  employed  in  the  breweries  of 
London  and  Edinburgh. 

The  yeasts  of  the  cerevisice  group  can  invert  cane  sugar,  select 
dextrose  from  Isevulose,  and  ferment  maltose,  but  they  cannot 
ferment  lactose,  nor  decompose  malto-dextrin. 

PASTORIANUS  GROUP. — These  are  wild  yeasts.  The  cells  are 
elongated  or  sausage-shaped,  and  six  or  eight  ascospores  are  pro- 
duced in  a  cell. 

8.  pastorianus  I. — A  bottom  fermentation  yeast  producing  a 
bitter  taste  in  beer. 

8.  pastorianus  II. — A  feeble  top  fermentation  form.  Surface 
cultures  on  yeast-water  gelatin  have  smooth  edges,  which  dis- 
tinguishes it  from  the  next  species. 

8.  pastorianus  III. — A  top  fermentation  form  producing  turbidity 
in  beer.  Surface  cultures  on  yeast-water  gelatin  have  woolly  margins. 

ELLIPSOIDEUS  GROUP. — These  are  wild  yeasts.  The  cells  are 
usually  ovoid,  or  pear-shaped,  sometimes  round,  rarely  elongated. 

Five  or  six  ascospores  are  produced  in  a  cell. 

8.  ellipsoideus  I. — A  bottom  fermentation  yeast  occurring  on 
ripe  grapes. 

S.  ellipsoideus  II. — A  bottom  fermentation  yeast  causing  turbidity 
in  beer. 

Both  the  pastorianus  and  ellipsoideus  groups  resemble  the  cerevisice 
group  in  their  chemical  actions,  but  they  are  able  in  addition  to 
decompose  malto-dextrin. 

8.  anomalus  is  a  yeast  forming  small  ovoid  cells.  It  is  curious 
in  that  the  spores  are  hemispheres  with  a  projecting  rim  at  the  base 
like  a  bowler  hat. 


468  A  MANUAL  OF  BACTERIOLOGY 

Another  point  in  the  identification  of  species  of  yeasts  is  the 
period  of  formation  of  films.  If  the  yeast  is  grown  in  wort  with 
free  access  of  air  and  is  undisturbed,  e.g.  in  a  beaker  capped  with 
filter-paper,  after  a  varying  period  a  film  composed  of  a  zooglosal 
mass  of  cells  appears  on  the  surface. 

If  yeast,  or  disintegrated  yeast-cells,  be  injected  into  animals, 
the  blood  acquires  specific  agglutinative  properties,  agglutinating 
the  yeast-cells  of  the  species  with  which  the  inoculation  has  been 
carried  out.1 

On  the  yeasts  of  fermentation,  see  Jorgensen,  Micro-organisms 
and  Fermentation,  4th  ed.,  1911  (C.  Griffin  and  Co.),  (full  bibliog.) 
Klocker,  Fermentation  Organisms. 

Examination  of  Yeasts 

The  yeasts  can  be  readily  examined  in  the  fresh  state  in  hanging- 
drop  preparations.  The  cells  should  be  young  or  they  will  not  be 
of  the  typical  form  ;  a  two  or  three  days'  old  culture  in  wort  or 
grape-sugar  solution  may  be  used.  The  yeasts  grow  well  at  20°- 
30°  C.  on  the  ordinary  gelatin,  agar,  and  potato,  but  wort  gelatin 
or  wort  agar  is  to  be  preferred.  The  elongated  cells,  common  to  all 
old  cultures  of  yeasts,  may  be  obtained  from  the  films  which  form 
on  wort  cultures  in  wide  flasks  or  beakers  after  two  or  three  weeks. 

In  order  to  stain  yeasts,  a  dilution  of  the  culture  should  be  made 
in  a  watch-glass  of  water,  so  that  the  cells  may  be  isolated,  as  they 
become  distorted  if  groups  form  in  the  preparations. 

If  the  yeast  has  been  grown  in  wort,  it  is  best,  before  staining, 
to  pour  off  the  fluid  from  the  deposit  of  cells  at  the  bottom  of  the 
flask  or  test-tube,  add  some  physiological  salt  solution  and  shake, 
then  allow  the  vessel  to  stand  for  an  hour  for  the  cells  to  sediment, 
or  centrifuge,  and  the  process  of  washing  may  be  repeated  once. 
Films  may  be  prepared  in  the  ordinary  way  and  stained  for  five 
minutes  in  Loffler's  methylene  blue,  washed  in  water,  dried,  and 
mounted.  Or  the  films,  after  air-drying,  may  be  fixed  by  immersion 
in  equal  parts  of  alcohol  and  ether  for  ten  minutes,  dried  in  the  air, 
and  stained  as  before.  The  preparations  can  also  be  stained  in 
gentian  violet  or  fuchsin,  or  by  Gram's  method. 

Ascospores  may  be  double  stained  by  preparing  films  of  a  sporing 
culture  in  the  ordinary  way,  staining  with  carbol-fuchsin  for  two 
minutes,  rinsing  in  water,  decolorising  with  5  per  cent,  sulphuric 
acid  and  with  alcohol,  rinsing  in  water,  counter -staining  with 
Loffler's  blue  for  five  minutes,  washing,  drying,  and  mounting. 
The  spores  are  red,  the  remainder  of  the  cells  blue. 

1  See  Macfadyen,  Centr.  f  Bakt.  (lte  Abt.);  xxx,  1901,  p.  368. 


CHAPTER  XVII 
THE  HYPHOMYCETES— ASPERGILLOSIS— RINGWORM 

The  Hyphomycetes 

THE  moulds  are,  for  convenience,  collectively  termed  the  Hypho- 
mycetes, but  this  is  not  a  strict  botanical  group.  They  are  Fungi 
having  as  a  common  character  a  plant  body  made  up  of  hyphse. 
They  are  multlccllular  individuals,  composed  of  filaments,  simple 
or  branched,  jointed  or  unjointed,  which  are  termed  hyphce,  and  are 
formed  by  the  end-to-end  union  of  elongated  cells.  When  the 
hyphaa  project  upwards  into  the  air  they  are  known  as  aerial 
hypha3,  and  when  downwards  into  the  fluid  or  medium  on  which 
the  organism  is  growing  as  submerged  hyphse,  and  the  compact 
tufts  or  masses  resulting  from  interlacing  hyphae  are  termed  mycelia. 
A  mycelium  may  form  a  hard  lignified  mass  or  pseudo-parenchyma, 
which  is  known  as  a  sclerotium,  such  as  is  met  with  in  ergot  and  in 
the  black  variety  of  mycetoma. 

Any  piece  of  the  mycelium  will  grow,  but  in  addition  moulds 
reproduce  by  multiple  spores,  which  may  be  asexual  or  sexual. 
Practically  all  moulds  produce  asexually  formed  spores  ;  some  pro- 
duce sexually  formed  spores  by  the  fusion  of  two  cells  or  gametes. 
The  two  principal  sexually  formed  spores  are  zygospores  and  asco- 
spores.  Zygospores  occur  in  Mucor  (see  p.  470).  In  ascospore 
formation,  after  conjugation  of  the  gametes,  instead  of  immediately 
developing  into  a  spore,  the  fertilised  cell  grows  into  a  mass  of  branch- 
ing hyphse,  some  of  the  cells  of  which  produce  spore  sacs  or  asci,  each 
of  which  contains  two  or  more  ascospores  (see  Penicillium,  p.  471). 

Asexual  spores  are  either  free,  borne  at  the  ends  or  sides  of  hyphae 
— conidia — as  in  Penicillium,  or  are  formed  in  specialised  spore 
cases — sporangia — as  in  Mucor. 

Usually  the  spore-bearing  hyphse  are  specially  differentiated,  and 
one  bearing  conidia  is  known  as  a  conidiophore,  one  bearing  a 
sporangium  as  a  sporangiophore.  Some  moulds  produce  spores  by 
segmentation  of  hyphee,  these  conidia  being  known  as  oidiat 

469 


470  A  MANUAL  OF  BACTERIOLOGY 

The  Fungi  are  divided  into  the  Phycomycetes,  Ascomycetes, 
Basidiomycetes,  and  Fungi  Imperfecti.  The  Phycomycetes  are 
distinguished  by  non-septate  or  slightly  septate  hyphae  and  zygo- 
spore-formation,  as  in  the  Mucors.  The  Ascomycetes  are  charac- 
terised by  the  development  of  the  cell  resulting  from  fertilisation 
into  cells,  some  of  which  become  spore  sacs  or  asci  containing  several 
spores.  Asexual  spores  are  usually  produced  as  well.  The  Basidio- 
mycetes have  spore-bearing  structures  known  as  basidia  ;  the  rusts, 
smuts,  toadstools,  puff-balls,  and  mushrooms  belong  to  this  group. 
All  fungi  which  do  not  fall  into  one  of  these  three  groups  are  placed 
among  the  Fungi  Imperfecti ;  most  of  them  probably  belong  to  the 
Ascomycetes.  Mucor  muiedo,  Penicillium  glaucum,  and  Asper- 
gillus  niger  may  be  taken  as  types  and  more  fully  described. 


Mucor  mucedo 

The  Mucoracice  belong  to  the  Phycomycetes,  and  are  divided  into 
some  eighteen  genera. 

Mucor  mucedo,  the  common  white  mould  which  appears  like 
tufts  of  cotton-wool  on  various  substances,  may  be  obtained  by 
exposing  some  moistened  bread  or  horse-dung  to  the  air  for  a  short 
time,  and  then  keeping  it  moist  under  a  bell- jar.  It  consists  of  a 
mycelium  composed  of  hyphae,  and  its  fluffy  appearance  is  caused 
by  aerial  hyphas.  The  aerial  hyphae  are  at  first  of  even  diameter 
throughout,  but  later  on  their  free  ends  become  swollen  and 
ultimately  form  spherical  bodies,  which  become  filled  with  spores, 
the  sporangia.  In  the  early  stage  the  whole  organism  forms  but 
a  single  cell,  the  protoplasm  of  which  is  granular  and  contains 
vacuoles  and  numerous  small  nuclei.  As  it  grows,  and  the  sporangia 
form,  these  become  separated  by  a  septum  from  the  hyphae,  and 
when  it  becomes  older  stil]  the  mycelial  hyphae  may  be  divided  into 
elongated  cells.  The  development  of  a  sporangium  takes  place  as 
follows  :  The  distal  end  of  an  aerial  hypha  swells,  and  immediately 
below  the  swollen  part  a  division  occurs  in  the  protoplasm  and  a 
cellulose  septum  is  formed,  so  that  the  swollen  part  is  separated 
off  from  the  rest  of  the  hypha,  forming  the  rudimentary  sporangium. 
The  sporangium  continues  to  grow,  and  its  protoplasm  undergoes 
multiple  fission  into  numerous  ovoid  masses,  the  spores,  each  of 
which  becomes  surrounded  with  a  cellulose  capsule.  The  septum 
separating  the  sporangium  from  the  hypha  projects  upwards  into 
the  interior  of  the  sporangium  as  a  club-shaped  knob  known  as  the 
columella.  When  the  sporangium  is  ripe  the  slightest  touch  causes 
its  wall  to  rupture,  so  liberating  the  spores.  When  placed  under 


PENICILLIUM  471 

favourable  conditions  the  spore  germinates,  and  the  buds  increase 
in  length  and  ultimately  form  hyphse. 

Occasionally  a  process  of  conjugation  occurs.  Two  adjacent 
hyphse  send  out  lateral  branches  which  come  in  contact  with  one 
another,  and  a  septum  forms  in  each,  separating  a  small  portion  of 
protoplasm  from  the  rest  of  the  hypha.  The  apposed  walls  of  the 
two  cells  become  absorbed  and  the  contents  mingle.  The  mass  of 
protoplasm  so  formed  becomes  surrounded  with  a  thick  cell-wall, 
giving  rise  to  an  inactive  spore-like  body,  the  zygospore,  which 
under  favourable  conditions  develops  like  an  ordinary  spore.  Some 
Mucors  form  thick-walled  resting  cells,  known  as  chlamydospores, 
in  the  vegetative  mycelium. 

Certain  Mucors  form  appreciable  amounts  of  alcohol  from  carbo- 
hydrates, and  M.  rouxii  has  been  used  for  the  commercial  production 
of  alcohol. 

Penicillium  glaucum 

Penicillium  belongs  to  the  Ascomycetes,  and  bears  conidiophores. 
Penicillium  glaucum  forms  the  bluish-green  mouldy  patches  familiar 
to  every  one.  It  is  by  far  the  commonest  of  all  species,  and  may 
be  obtained  from  moist  bread  or  jam  or  by  exposing  a  gelatin  plate 
to  the  air  for  a  short  time.  If  the  mouldy  patch  be  rubbed  a  fine 
greenish  dust  comes  away.  This  dust  consists  of  myriads  of  spores  ; 
if  a  little  of  it  be  transferred  with  a  moistened  needle  to  a  gelatin 
plate,  or,  better  still,  to  a  hanging-drop  preparation,  the  growth  of 
the  organism  can  be  studied.  After  two  or  three  days  little  white 
specks  will  be  observed,  which  microscopically  are  found  to  consist 
of  tufts  of  delicate  interlacing  hyphse  ;  these,  becoming  interwoven, 
ultimately  form  a  tough  mycelium.  The  patches  of  growth  are 
circular,  and  the  hyphse  will  be  found  to  radiate  from  the  centre. 
As  the  patch  increases  in  size  it  changes  in  colour,  becoming  bluish- 
green,  though  the  margin  for  some  time  still  remains  white.  From 
the  upper  surface  of  the  mycelium  delicate  aerial  hyphse  grow 
upwards,  and  from  the  under  surface  short  submerged  ones  project 
downwards. 

The  hyphse  are  composed  of  elongated  cells  arranged  end  to  end, 
the  cell-walls  of  which  consist  of  cellulose  enclosing  a  more  or  less 
vacuolated  protoplasm  containing  several  nuclei. 

The  aerial  hyphse  are  unbranched  filaments,  but  as  development 
proceeds  the  distal  ends  branch  dichotomously,  the  branches 
remaining  short  and  nearly  parallel  to  one  another,  so  that  a  kind 
of  brush  is  produced.  The  ultimate  branches  are  known  as  sterig- 
mata.  The  ends  of  the  sterigmata  become  constricted  so  that  little 


472  A  MANUAL  OF  BACTERIOLOGY 

globular  masses,  the  spores,  are  formed  ;  this  process  is  repeated 
until  a  chain  of  spores  results,  the  proximal  one  being  the  youngest. 
A  spore  when  placed  under  favourable  conditions  germinates,  a  little 
bud  appearing,  elongating,  and  forming  a  hypha,  just  as  in  Mucor. 
Brefeld,  by  sowing  spores  on  moist  bread,  inverting  the  bread, 
and  examining  at  intervals,  observed  a  sexual  method  of  repro- 
duction in  Penicillium.  Two  sets  of  spiral  cells  develop  on  a  thick 
hypha,  they  intertwine,  their  contents  probably  mingle,  and  from 
the  union  or  carpogonium  a  tube-like  hypha  develops,  which 
becomes  surrounded  and  enclosed  by  branching  hyphse  from  the 
mother  cell.  By  further  development  and  thickening  of  the  cell- 
walls  a  sclerotium  forms  ;  it  is  a  hard  solid  body,  yellowish  in  colour, 
and  resembles  a  grain  of  sand,  the  carpogonium  being  at  the  centre. 
If  placed  in  favourable  conditions  the  sclerotia  germinate  after  some 
time.  Two  forms  of  hyphse  are  produced,  one  thick,  the  other 
thin  ;  the  latter  become  much  twisted.  The  thick  hyphce  become 
branched,  and  ultimately  a  number  of  pear-shaped  bodies  are  pro- 
duced. The  contents  of  these  bodies  then  become  broken  up  and 
form  spores  ;  the  bodies  are  known  as  asci  and  the  spores  as  asco- 
spores.  From  the  ascospores  the  ordinary  mycelial  form  again 
develops.1 

Aspergillus  niger 

Aspergillus  also  belongs  to  the  Ascomycetes,  and  representatives 
of  this  genus  are  common  on  damp  and  decaying  vegetable  matter. 
The  asci  occur  as  golden-yellow  bodies  in  the  mycelium.  It  forms 
conidiophores  which  are  unbranched  and  are  swollen  at  the  tip. 
Short  unbranched  stalks  (sterigmata)  grow  on  this  swelling  and  on 
the  tips  ot  these  the  spores  develop.  A  process  of  sexual  reproduc- 
tion occurs  very  like  the  one  observed  in  Penicillium.  Aspergillus 
niger  grows  well  on  the  ordinary  laboratory  media,  producing  on 
potato  a  powdery,  sooty  growth  after  a  time.  Aspergillus  gla.ucus 
is  a  common  green-spored  species. 

With  the  exception  of  the  ringworm  and  allied  fungi, 
which  produce  parasitic  skin  affections,  the  Hyphomycetes 
are  not  of  very  great  pathological  importance.  In  the 
ear  and  nose  mucors  and  aspergilli  may  be  met  with,  but 
in  these  situations  they  are  epiphytes  rather  than  parasites, 
and  the  same  species  occur  in  bronchiectases  and  pulmonary 

1  See  Brefeld,  Quart.  Journ.  Microscop.  Soc.,  vol.  xv,  p.  342. 


SPOROTRICHOSIS  473 

vomicge.  Occasionally,  however,  a  pneumono-mycosis 
has  been  met  with,  the  mycelium  of  the  fungus  ramifying 
in  the  lung  tissue  and  setting  up  irritative  and  other 
changes.  "  Pneumono-mycosis  "  or  "  pulmonary  asper- 
gillosis  "  is  especially  a  trade  disease  among  bird-rearers. 
Grain  is  taken  into  the  mouth  and  the  bird  is  fed  with  it, 
and  in  the  course  of  this  operation  the  mould  spores  are 
inhaled.  The  course  of  the  disease  is  much  like  chronic 
bronchitis  or  pulmonary  tuberculosis.  The  species  met 
with  in  this  condition  seems  generally  to  have  been  the 
Aspergillus  fumigatus. 

The  black  variety  of  madura  disease,  as  already  stated 
(p.  459),  is  due  to  a  fungus  form,  and  varieties  of  mycetoma 
may  be  caused  by  fungi  belonging  to  Aspergillus. 

Sporotrichosis  l 

A  rare  disease  clinically  resembling  syphilis  or  tuber- 
culosis, characterised  by  indurated  granulomata  like 
gummata,  which  subsequently  break  down,  suppurate  and 
ulcerate.  Potassium  iodide  has  a  curative  action  on  the 
condition. 

In  the  pus  of  the  lesions  large  ovoid  refractile  bodies 
suggestive  of  yeasts  or  of  large  spores  may  be  detected, 
but  no  mycelium. 

Cultures  are  best  obtained  on  maltose  agar  (p.  477) 
from  non-ulcerated  lesions  ;  agar  and  potato  may  also 
yield  growths.  The  organism  (Sporotrichon  Beurmanni) 
grows  as  small  raised  woolly  colonies,,  at  first  white,  after- 
wards becoming  brown.  The  growths  consist  of  a  felted 
mycelium  of  filaments  with  spores  and  yeast-like  cells. 
It  produces  granulomata  in  inoculated  mice.  The  botanical 
position  of  the  organism  is  uncertain  ;  by  some  it  is  regarded 

1  See  Walker  and  Ritchie,  Brit.  Med.  Journ.,  1911,  vol.  ii,  p.  1  ; 
Gougerot,  Journ.  of  State  Med.,  xxi,  1913,  p.  614  et  seq. 


474  A  MANUAL  OF  BACTERIOLOGY 

as  a  true  fungus.  It  is  stated  to  occur  on  decaying  vege- 
table matter  and  to  be  the  cause  of  epizootic  lymphangitis 
in  the  horse — a  disease  having  a  superficial  resemblance  to 
farcy — in  the  pus  of  which  oat-shaped  bodies  are  found, 
the  "  cryptococcus  "  of  Rivolta. 

Thrush 

Thrush  is  due  to  an  organism  (O'idium  or  Monilia  albi- 
cans)  which  is  usually  classed  among  the  Hyphomycetes. 
It  forms  the  whitish  patches  so  frequently  seen  on  the 
mucous  membrane  of  the  mouth  and  pharynx  in  children 
and  in  those  suffering  from  wasting  diseases  but  a  general 
infection  has  occasionally  been  produced  by  it.  If  one 
of  these  patches  is  removed  and  teased  up,  it  will  be  found 
to  consist  of  masses  of  tangled  mycelial  threads  with  yeast- 
like  budding.  The  organism  can  be  readily  cultivated  on 
all  the  ordinary  laboratory  media,  and  will  also  grow  on 
slightly  acid  media  such  as  wort  gelatin.  It  produces 
whitish,  membranous,  adherent  growths,  in  which  it 
appears  morphologically  under  two  forms — as  masses  of 
tangled  filaments  or  hyphae  and  as  yeast-like  cells.  On 
aoid  media  the  latter  exclusively  occur,  on  alkaline  the 
former  predominate.  It  liquefies  gelatin,  stains  by  Gram's 
method,  produces  an  alkaline  reaction  by  the  formation 
of  ammonium  carbonate,  and  does  not  ferment  lactose. 
Inoculated  on  to  a  damaged  mucous  membrane  the 
"  thrush  "  patches  appear,  subcutaneously  it  produces  an 
abscess,  and  injected  into  the  peritoneum  a  general  infec- 
tion, followed  by  death  and  accompanied  by  a  sero-purulent 
peritonitis. 

Cultivation  and  Examination 

The  Hyphomycetes  can  be  cultivated  on  the  ordinary  laboratory 
media,  but  wort-agar,  or  wort -gelatin,  potato,  bread,  or  maltose  agar 
is  to  be  preferred. 


RINGWORM  475 

They  can  be  examined  by  removing  a  portion  of  the  growth, 
teasing  up  gently  with  needles  in  a  little  50  per  cent,  alcohol  con- 
taining a  trace  of  ammonia,  removing  the  surplus  fluid  with  blotting- 
paper,  and  mounting  in  Farrant's  solution  or  in  glycerine  jelly.* 
If  desired,  they  may  be  stained  by  the  irrigation  method  with  f  uchsin. 
Thrush  may  be  examined  in  this  way. 

In  the  tissues  they  may  be  stained  with  hsematoxylin  or  methylene 
blue,  or  by  Gram's  or  by  Weigert's  method. 

Ringworm 

The  ringworm  fungi  must  probably  be  included  in  the 
group  of  the  Hyphomycetes.  Human  ringworm,  formerly 
regarded  as  a  single  disease,  has  been  proved  to  comprise 
at  least  two  affections  through  the  researches  of  Sabouraud. 
These  two  forms  are  distinguished  from  each  other  clini- 
cally and  by  differences  in  the  parasitic  organisms. 

The  first  variety  is  an  affection  of  early  childhood, 
forming  80  to  90  per  cent,  of  the  ringworms  met  with  in 
London  ;  it  never  attacks  the  scalp  of  adults,  never  affects 
the  beard  or  nails,  is  very  intractable,  and  frequently 
epidemic.  The  parasite  is  characterised  by  small  round 
or  ovoid  spores  measuring  3  /x  to  4  /x  in  diameter.  Affected 
hairs  are  generally  broken  off,  forming  relatively  long 
stumps,  greyish  in  colour,  and  possessing  a  whitish  sheath. 
When  suitably  prepared  in  potash  this  sheath  is  seen  to 
be  composed  of  the  spores  agglomerated  together  without 
apparent  order,  and  the  hairs  themselves  are  filled  with 
delicate  parallel  mycelial  threads  (Fig.  52).  The  fungus  is 
named  the  Microsporon  Audouini. 

The  second  variety  comprises  the  ringworms  with  large 
spores,  and  is  divided  into  two  groups  by  Sabouraud.  The 
first  of  these  groups  is  exclusively  of  human  origin,  and 
has  a  marked  tendency  to  affect  the  interior  of  the  hairs 
only,  and  hence  the  organism  has  been  termed  the  Tricho- 
phyton  megalosporon  endothrix.  The  other  group  is  of 
animal  origin,  and  the  spores  are  met  with  chiefly  on  the 


476 


A  MANUAL  OF  BACTERIOLOGY 


outside  of  the  hairs,  and  the  fungus  is  hence  termed  the 
Trichopliyton  megalosporon  ectothrix. 

9  The  endothrix  form  occurs  later  in  childhood,  is  not  so 
persistent  as  the  Microsporon,  and  does  not  attack  the 
nails  or  beard.  Microscopically,  the  fungus  is  seen  to 
consist  of  beaded  threads,  which  are  rounded  or  ovoid 
spores  arranged  end  to  end.  The  ectothrix  form  rarely 


FIG.  52. — Ringworm  in  a  hair.      X  350. 

attacks  the  scalp,  but  is  responsible  for  all  the  tinea  sycosis 
and  ringworm  of  the  nails  and  half  the  cases  of  tinea 
circinata.  Suppuration  is  common  in  this  form.  Micro- 
scopically appearances  differ ;  generally  the  spores  are 
arranged  in  chains,  but  the  sporulation  is  less  regular  than 
in  the  endothrix.  The  spores  in  the  endothrix  and  ectothrix 
varieties  measure  4  /m  to  12  JUL  in  diameter. 

The  ringworm  fungi  can  be  readily  cultivated  on  all 
the  ordinary  media — beer- wort  agar  and  beer- wort  gelatin 
being  especially  favourable.  They  form  whitish  fluffy 
growths  with  rapid  liquefaction  of  gelatin.  In  order  to 
obtain  cultivations  the  diseased  hairs  or  stumps  are 


RINGWORM  477 

removed  by  forceps  and  placed  on  a  sterile  glass  slide. 
The  aerial  portion  of  the  hair  is  then  cut  away  by  means 
of  a  sterile  scalpel,  and  the  diseased  portion  is  divided  into 
small  fragments.  These  can  be  picked  up  with  a  moistened 
platinum  needle  and  transferred  to  the  culture  media, 
preferably  beer-wort  agar.  In  some  cases  a  pure  culture 
is  thus  obtained,  but  in  others  further  treatment  is  neces- 
sary. When  the  Trichophyton  or  Microsporon  has  thrown 
up  its  aerial  hyphaa  the  plug  of  wool  is  removed  from  the 
tube  and  the  mouth  well  flamed  ;  the  tube  is  then  held 
inverted  over  a  Petri  dish  containing  solidified  maltose 
agar.  A  sharp  tap  or  two  is  given  to  the  tube,  sufficient 
to  cause  the  spores  to  drop,  and  the  dish  is  re-covered. 
A  growth  of  the  organism  from  single  isolated  spores  thus 
ensues,  and  pure  cultures  can  be  obtained  (Blaxall). 

The  various  forms  of  the  ringworm  fungi  can  be  differen- 
tiated by  cultures,  but  it  is  necessary  when  comparing  them 
to  employ  media  of  identical  composition,  because  slight 
differences  in  the  latter  are  liable  to  induce  marked  changes 
in  the  characters  of  the  cultures.  A  favourite  medium, 
used  by  Sabouraud  and  by  Blaxall,  is  maltose  agar  : 

Peptone 0*5  grm. 

Maltose 3-8  grm. 

Agar-agar 1-3  grm. 

Water 100  c.c. 

Blaxall  found  that  different  samples  of  maltose  materially 
influenced  the  characters  of  the  cultures. 

Characters  of  the  cultures. — Cultures  are  incubated  at 
30°  C.  The  colonies  of  the  Microsporon  do  not  show  any 
growth  until  about  the  seventh  day ;  little  white  downy 
tufts  then  appear.  The  fully  developed  growth  on  maltose 
agar  forms  a  large  white  downy  patch  with  a  small  central 
boss  ;  on  potato  white  downy  patches  appear  with  brown 
discoloration. 


478  A  MANUAL  OF  BACTERIOLOGY 

The  endothrix  variety  commences  to  grow  in  six  or 
seven  days,  and  on  maltose  agar  in  about  a  month  forms 
a  rounded  patch  with  a  central  crateriform  depression, 
the  whole  being  dusted  with  fine  white  powder  (Fig.  53)  ; 
on  potato,  powdery  stars  develop  tinged  with  yellow  and 
usually  without  discoloration  of  the  medium. 

The  cultures  of  the  ectothrix  form  are  variable.  They 
commence  on  the  third  or  fourth  day  ;  some  develop 


FIG.  53. — Culture  of  the  ringworm  organism.     Endothrix  form. 

whitish  smooth,  or  wrinkled  growths  ;  others,  from  the 
dog,  form  dry,  brown,  wrinkled,  powdery  growths  ;  others, 
of  bird  origin,  form  purplish  growths. 

Microscopically,  all  the  fungi  show  masses  of  mycelial 
threads  with  spores.  They  stain  with  the  ordinary  anilin 
dyes  and  also  by  Gram's  method,  and  can  be  mounted  in 
glycerin  jelly  in  the  manner  described  at  p.  475. 

Macfadyen  found  that  the  ringworm  organism  produces 
an  active  peptonising  enzyme,  and  seems  to  increase  the 
solubility  of  keratin  when  grown  on  it ;  no  inverting 
enzyme  could  be  isolated. 


RINGWORM  479 


Clinical  Examination 

The  hairs  should  be  treated  first  with  ether  and  then  with  caustic 
potash  solution  of  about  7  per  cent,  strength.  In  this  reagent  they 
may  remain  for  from  a  few  hours  to  a  few  days  ;  they  are  then 
floated  on  to  a  slide  and  carefully  covered  with  a  cover-glass. 
Permanent  preparations  may  be  mounted  in  Farrant's  solution  or 
in  glycerine  jelly. 

Hairs,  after  treatment  with  ether,  may  be  stained  by  the  following 
method : 

(1 )  Stain  in  anilin-gentian  violet  for  one  to  two  minutes,  and  blot, 

(2)  Treat  with  Gram's  iodine  solution  for  one  to  two  minutes, 
and  blot. 

(3)  Decolorise  carefully  (watching  microscopically)  with  anilin 
oil  containing  1  per  cent,  of  hydrochloric  acid. 

(4)  Treat  with  anilin  oil  and  then  with  anilin  oil  and  xylol. 

(5)  Clear  in  xylol,  and  mount  in  Canada  balsam. 
ERYTHRASMA. — Due   to  infection  with  a  fungus   (Microsporon 

minutissimum),  very  difficult  to  cultivate,  which  occurs  as  extremely 
long,  fine  filaments. 

FAVUS. — Favus  is  due  to  a  fungus  discovered  by  Schoenlein  in 
1839 — the  Achiorion  Schoenleinii.  It  is  seen  as  a  mycelial  growth 
with  spores  in  the  patches.  The  organism  grows  well  on  maltose 
agar,  forming  fluffy,  woolly,  moss-like  colonies  with  radiating  out- 
growths, first  grey  and  then  yellowish.  It  occurs  on  mice  and  other 
animals. 

DHOBIE  ITCH. — Caste! lani  has  isolated  three  trichophyton-like 
organisms  in  this  disease. 

PITYRIASIS  ALBA. — In  this  disease  Unna's  "  bottle  bacillus  "  is 
invariably  present.  It  occurs  as  large  round  or  oval  bodies  like 
yeast-cells,  which  may  occasionally  show  budding. 

PITYRIASIS  VERSICOLOR. — In  the  epidermal  scales  of  this  skin 
affection  a  fungoid  organism  (Microsporon  furfur)  is  present.  It 
occurs  as  short  and  thick  curved  hyphse  between  which  are  masses 
of  large  coarse  spores.  It  has  not  been  cultivated  (or  very  rarely). 

PINTA. — A  skin  disease  met  with  in  South  America.  In  the  scales 
short  mycelial  filaments  with  large  (8-12  ^u)  spores  are  seen.  Various 
organisms  have  been  cultivated  belonging  to  the  genera  PeniciHium 
and  Aspergillus. 

PIEDRA. — A  disease  of  the  hairs  met  with  in  South  America. 
The  nodosities  on  the  hairs  are  composed  of  masses  of  very  large 
refractile  spores. 


CHAPTER  XVIII 
THE  PROTOZOA  x 

The  General  Structure  of  the  Protozoa — Pathogenic  Amoebae 
—  Trypanosomata  —  Leishman-Donovan  Body  —  Spirochaetac — 
Coccidia — Malaria 

THE  Protozoa  are  an  important  group  of  unicellular  organisms, 
regarded  as  animal  in  nature,  and  sharply  and  definitely  distin- 
guished from  the  rest  of  the  animal  kingdom,  to  which  the  names 
of  metazoa  and  enterozoa  are  applied.  The  latter  consists  of  many 
cells,  differentiated  to  perform  different  functions,  and  arranged  in 
two  layers — endoderm  and  ectoderm — around  a  central  cavity,  the 
enteron. 

"  It  is  true  that  some  protozoa  consist  of  aggregates  of  cells,  and 
should  therefore  be  entitled  to  be  called  multicellular  ;  yet  an 
examination  of  the  details  of  structure  of  these  cell -aggregates  and 
of  their  life-history  establishes  the  fact  that  the  cohesion  of  the  cells 
in  these  instances  is  not  an  essential  feature  of  the  life  of  such  multi- 
cellular  protozoa,  but  a  secondary  and  non-essential  arrangement. 
Like  the  budded  '  persons  '  forming,  when  coherent  to  each  other, 
undifferentiated  '  colonies '  among  the  polyps  and  corals,  the 
coherent  cells  of  a  compound  protozoon  can  be  separated  from  one 
another  and  live  independently  ;  their  cohesion  has  no  economic 
significance.  Each  cell  is  precisely  the  counterpart  of  its  neighbour  ; 
there  is  no  common  life,  no  distribution  of  function  among  special 
groups  of  the  associated  cells,  and  no  corresponding  differentiation 
of  structure.  As  a  contrast  to  this,  we  find  in  the  simplest  enterozoa 
that  the  cells  are  functionally  and  structurally  distinguishable  into 
two  groups — those  which  line  the  enteron  or  digestive  cavity,  and 
those  which  form  the  outer  body  wall.  The  cells  of  these  two  layers 

1  See  Lankester's  Treatise  on  Zoology,  Part  I,  first  and  second 
Fascicles,  1907  and  1909  ;  Minchin  in'  Clifford  Allbutt's  System  of 
Medicine,  ed.  2,  vol.  ii,  pt.  ii ;  Hartog  in  Cambridge  Natural  History, 
vol.  i. 

480 


SARKODINA  481 

are  not  interchangeable,  but  are  fundamentally  different  in  proper- 
ties and  structure  "  (Ray  Lankester).  It  is  true  that  in  some 
instances  there  may  be  a  difficulty  in  deciding  whether  an  organism 
is  vegetable  or  animal,  and  Haeckel  proposed  to  include  all 
indeterminate  unicellular  organisms  in  a  distinct  kingdom,  the 
Protista. 

The  cytoplasm  of  a  protozoon  is  commonly  differentiated  into 
an  outer,  clearer,  denser  layer  or  ectosarc,  and  an  inner,  granular, 
more  fluid  portion,  the  endosarc.  The  cytoplasm  is  sometimes 
naked,  or  may  be  covered  with  a  cuticle,  usually  protein  in  nature. 
The  cytoplasm  contains  a  well -marked  nucleus,  sometimes  a 
secondary  nucleus,  and  occasionally  subsidiary  chromatin  particles 
or  chromidia.  A  contractile  vacuole,  which  is  an  excretory  organ, 
is  frequently  present. 

In  most  protozoa  reproduction  takes  place  by  simple  division  or 
fission,  and  by  a  process  of  spore-formation  ;  in  others  reproduction 
is  exclusively  by  spores,  which  are  often  formed  by  a  complicated 
process  of  development.  In  many  of  the  protozoa  a  simple  form 
of  sexual  reproduction  by  conjugation  occurs.  Two  dissimilar 
cells  (gametes)  are  produced,  the  larger  comparable  to  female  cells 
or  ova  and  termed  macrogametes,  the  smaller  comparable  to  male 
elements  or  spermatozoa  and  termed  microgametes.  The  cells 
from  which  the  gametes  are  derived  are  known  as  gametocytes.  The 
gametes  conjugate  and  form  a  zygote,  which  usually  divides  into 
a  number  of  spores  from  which  the  adult  is  reproduced. 

In  certain  cases  sexually  differentiated  individuals  reproduce 
by  fission  without  conjugation  ;  this  phenomenon  is  termed  parthe- 
nogenesis. 

Various  classifications  of  the  Protozoa  have  been  suggested. 
Biitschli  divides  them  into  four  classes  :  I.  The  Sarkodina  (p.  481)  ; 
II.  The  Mastigophora  (p.  487) ;  III.  The  Infusoria  (p.  507) ;  and 
IV.  The  Sporozoa  (p.  508). 


Class  I. — Sarkodina 

There  are  Protozoa  in  which  the  cell  protoplasm  is  naked,  and 
locomotion  and  ingestion  of  food  are  performed  by  means  of  tem- 
porary protoplasmic  processes  or  pseudopodia. 

The  Sarkodina  includes  a  number  of  forms  of  very  varied  mor- 
phology and  habits,  such  as  the  Amoebae,  Heliozoa,  Radiolaria,  and 
Foraminifera,  the  three  latter  groups  being  characterised  by  the 
presence  of  a  siliceous  or  calcareous  skeleton  or  shell. 

31 


482  A  MANUAL  OF  BACTERIOLOGY 

Pathogenic  Amoebae  l 

Three  species  of  Amcebce  seem  to  be  parasitic  in  man, 
and  the  generic  name  of  Entamceba  has  been  given  to 
them.  One,  the  E.  buccalis,  occurs  in  the  mouth  in  dental 
caries,  the  other  two  inhabit  the  intestine.  One  of  the 
latter,  the  Entamceba  coli  (Amoeba  coli,  Losch),  occurs  in 
the  upper  part  of  the  large  intestine  and  appears  to  be 


FIG.  54. — Amceba  histolytica.     (After  Councilman  and  Lafleur.) 

harmless  ;  the  other,  the  Entamceba  histolytica,  is  regarded 
as  the  cause  of  amoebic  or  tropical  dysentery. 

The  Entamceba  histolytica  is  met  with  in  the  faeces  in 
these  cases,  and  also  in  the  pus  of  the  so-called  tropical 
abscess  of  the  liver.  It  is  especially  abundant  in  the 
mucoid  material  during  the  acute  stage.  The  E.  histoly- 
tica is  a  large  protoplasmic  mass  measuring  25  to  35  /x.  in 
diameter,  possessed  of  slow  amoeboid  movement,  and 
having  a  clearer  outer  zone  or  ectosarc  and  a  granular 
endosarc.  The  pseudopodia  are  always  blunt,  never 

1  Councilman  and  Lafleur,  Johns  Hopkins  Hosp.  Reps.,  vol.  ii,  1891  ; 
Schaudinn,  A.K.  Gesundheitsamte,  xix,  p.  547 ;  Strong,  Musgrave, 
Clegg,  Thomas  and  Woolley,  Bureau  of  Gov.  Laboratories,  Manila 
Bulls.  18  and  32. 


PATHOGENIC  AMOEBA  483 

pointed  (Fig.  55).  Tn  the  endosarc  highly  refractile  granules 
occur,  and  it  often  contains  blood- corpuscles  and  a  vacuole 
(Fig.  54,  6).  A  nucleus  can  also  be  demonstrated,  but 
being  poor  in  chromatin,  it  stains  with  difficulty  (Fig.  54,  a). 
According  to  Schaudinn,  the  E.  coli  differs  from  the  E. 
histolytica  in  that  the  ectoplasm  is  not  distinctly  seen 
except  during  the  formation  of  a  pseudopodium,  and  the 


FIG.  55. — Changes  in  form  of  an  Amosba  histolytica  observed  on 
a  warm  stage,  and  drawn  at  intervals  of  one  minute.  (Semi- 
diagrammatic  by  the  writer.) 

nucleus  stains  deeply.  The  development  of  the  two  forma 
is  also  different.  E.  coli  multiplies  by  simple  binary  fission, 
and  also  by  multiple  fission  into  eight  small  amoebae. 
Encystment  may  also  occur,  with  repeated  binary  division 
of  nucleus  and  protoplasm,  part  of  the  nucleus  being  cast 
off  and  ultimately  the  cyst  contains  eight  nuclei  around 
which  the  protoplasm  collects,  so  that,  if  swallowed,  eight 
small  amoebae  are  set  free. 

The  E.  histolytica  multiplies  by  binary  fission,  and  also 
by  irregular  gemmation,  so  that  an  indefinite  number  of 


484  A  MANUAL  OF  BACTERIOLOGY 

small  amoebae  is  formed.  Instead  of  encystment,  as  in 
the  E.  coli,  resistant  spores  are  formed.  The  nucleus  gives 
off  chromidia,  some  of  which,  together  with  portions  of  the 
ectoplasm,  are  extruded  and  become  spores  surrounded 
by  tough  capsules.  Infection  of  a  fresh  host  apparently 
occurs  only  with  material  containing  these  spores. 

The  presence  of  the  amoeba  in  the  pus,  and  especially 
in  the  walls,  of  tropical  abscesses  is  of  considerable  diag- 
nostic significance,  and  the  parasite  is  considered  to  be 
the  etiological  agent  in  amoebic  or  tropical  dysentery 
(see  "  Dysentery  ").  The  amoebae  are  not  usually  observed 
in  the  abscess  pus  at  the  time  of  operation,  but  make 
their  appearance  in  the  discharge  about  the  third  day, 
i.e.  when  the  wall  of  the  abscess- cavity  is  contracting. 
In  the  true  tropical  abscess  the  ordinary  pyogenic  organ- 
isms are  absent,  unless  a  secondary  infection  has  occurred, 
which  is  the  exception.  The  abscess  is  usually  single,  and 
Rogers  suggests  that  the  amoebae  reach  the  liver  through 
adhesions  between  it  and  the  bowel.  The  amoebae  may 
be  cultivated  on  ordinary  or  on  water  agar  provided  some 
bacterium  is  present  at  the  same  time,  e.g.  B.  coli,  cholera 
vibrio,  etc.  Material  rich  in  amoebae  may  be  smeared 
over  agar  plates,  which  are  grown  at  25°-30°  C.  for  twenty- 
four  to  forty- eight  hours,  and  are  then  examined  with  a 
low  power.  At  any  spot  where  isolated  amoebae  are 
observed,  with  a  little  dexterity  the  organism  may  be 
lifted  up  with  a  fine  needle  and  transferred  to  a  fresh 
plate,  and  by  a  repetition  of  the  process  pure  cultures 
may  be  obtained.  The  cultivated  amoebae  are  pathogenic 
for  monkeys,  and  induce  abscess  on  inoculation  into  the 
liver.  Musgrave  and  Clegg  (loc.  cit.)  are  of  opinion  that 
all  amoebae  are,  or  may  become,  pathogenic. 


PATHOGENIC  AMOEBA  485 


Clinical  Diagnosis 

1.  A   drop  of  the   dysenteric  discharge  (the  mucous  portions 
should  be  chosen  from  the  stools),  pus,  or,  better,  a  scraping  from 
the  wall  of  the  abscess,  diluted,  if  necessary,  with  a  little  warm 
(37°  C.)  physiological  salt  solution,  is  placed  on  a  slide,  covered 
with  a  cover-glass,  and  examined  microscopically  with  a  j-  or  J-inch 
objective.     The  amoebae  will  be  readily  recognised,  and  may  be 
examined  more  critically  with  a  ^L-irich  oil-immersion.     To   be 
certain  that  the  bodies  are  amoebae,  the  amoeboid  movements  must 
be  observed  by  keeping  the  preparation  on  a  warm  stage. 

The  stools  should  be  fresh,  unmixed  with  urine,  collected  in  a 
warmed  bed-pan,  and  kept  at  blood-heat  until  examined,  which 
should  be  done  as  soon  as  possible. 

2.  The  living  amoebae  in  the  stools  may  be  stained  by  the  irriga- 
tion method  with  a  weak  (|-1  per  cent.)  aqueous  solution  of  neutral 
red.     Preparations  may  also  be  stained  by  irrigation  with  methyl- 
ene-blue   and  Beale's  carmine  ;    the  latter  stains  the  nucleus,  the 
former  does  not.     The  preparation  may  be  rendered  permanent 
by  washing  away  the  excess  of  stain,  and  running  in  some  50  per  cent, 
glycerin  by  irrigation. 

3.  Probably  Heidenhain's  iron-haematoxylin  method  is  the  best 
for  staining  this  and  other  protozoa  : 

(a)  Make  smears  of  the  material  and  drop  while  wet  into  the 
fixative — two  parts  of  saturated  aqueous  mercuric  chloride  solution, 
one  part  of  alcohol,  with  a  few  drops  of  glacial  acetic.  They  remain 
in  this  for  ten  minutes. 

(6)  Wash  in  weak  spirit  and  then  in  weak  spirit  coloured  with 
iodine,  and  finally  wash  in  distilled  water. 

(c)  Treat  with  4  per  cent,  iron-alum  solution  for  six  to  ten  hours. 

(d)  Stain  in  Heidenhain's  hsematoxylin  for  at  least  six  hours. 

(e)  Differentiate  in  1  per  cent,  iron-alum,  watching  microscopi- 
cally. 

(/)  Wash  well  in  tap-water,  pass  through  alcohol  and  xylol,  and 
mount. 

4.  Twort's  stain  may  be  used  for  sections.  .  The  stain  (which  is 
a  compound  neutral  red  and  light  green  preparation)  is  best  made 
by  rubbing  up  0-25  grm.  of  the  stain  (Griibler's)  with  some  clean 
sharp  sand  in  a  mortar  ;  this  prevents  the  stain  going  into  a  sticky 
mass  when  the  alcohol  is  added.     To  the  powder  so  obtained  is 
now  added  some  purest  methyl  alcohol  (Merck's),  acetone-free, 
containing  5  per  cent,  by  volume  of  glycerin.     Rub  up  well  to 


486  A  MANUAL  OF  BACTERIOLOGY 

obtain  a  saturated  solution  ;  then  pour  off  and  add  a  further 
quantity  of  alcohol -glycerin  solution,  and  repeat  the  trituration  ; 
about  100  c.c.  stain  can  be  made  from  the  quantity  given. 

The  solution,  when  filtered,  should  be  kept  in  a  well-stoppered 
bottle  (and  if  a  completely  saturated  solution  has  been  obtained, 
add  10  per  cent,  more  alcohol-glycerin  mixture).  The  stain  may 
be  purchased  ready  for  use. 

Tissues  to  be  examined  should  be  fixed  in  Miiller's  fluid  con- 
taining 10  per  cent,  of  formalin,  but  on  no  account  should  10  per 
cent,  formalin  alone  be  used. 

Paraffin  sections  (after  xylol,  alcohol  and  distilled  water)  are 
stained  for  about  five  minutes  with  the  stain  made  up  by  mixing 
one  part  of  distilled  water  with  two  parts  of  the  glycerin-alcohol 
stain  solution.  Sometimes  in  staining  such  organisms  as  glanders 
ten  minutes  may  be  necessary,  especially  if  insufficient  stain  is  in 
solution  and  the  room  temperature  is  low.  Rinse  in  distilled 
water. 

Fix  for  half  to  one  minute  in  Unna's  glycerin-ether  mixture — 
2  per  cent,  in  distilled  water.  Rinse  in  distilled  water. 

Differentiate  and  dehydrate  in  absolute  alcohol.  Should  there 
be  much  precipitate,  this  can  easily  be  removed  by  a  few  drops 
of  methyl  alcohol,  or  better  by  a  mixture  of  equal  parts  of  absolute 
alcohol  and  xylol.  Pass  through  xylol  and  mount. 

Various  elements  stain  different  colours,  viz.  chromatin  of  nuclei, 
purple  red  ;  mucoid  and  colloid  degenerations,  bright  orange  red  ; 
fetal  cartilage,  orange  red  ;  fibrous  tissue,  blue-green  ;  erythrocytes, 
light  grass -green.  Micro-organisms  stain  bright  red  and  stand  out 
in  marked  contrast  to  the  green  connective  tissue  containing 
them. 

Animal  parasites,  e.g.  amcebaa,  also  stain  well.  The  stain  has 
the  advantage  of  leaving  all  the  tissues  sharply  differentiated. 

Allusion  may  here  be  made  to  the  Mycetozoa  (Myxomycetes). 
These  are  masses  of  protoplasm  resembling  huge  amoebae,  which 
are  found  on  decaying  vegetable  matter.  By  some  they  are  regarded 
as  vegetable,  by  others  as  animal,  in  nature,  and  belonging  to  the 
Amoebae  of  the  Sarkodina.1  Some  important  plant  diseases,  such 
as  the  "  finger-and-toe  "  of  cabbage  roots,  are  due  to  their  activity. 
The  finger-and-toe  disease  is  due  to  an  amoaboid  parasite  (Plasmo- 
diophora  brassicce,  by  some  included  among  the  Amcebce),  the  cycle 
of  which  begins  with  spores  from  which  small  flagellulae  are  set 
free.  Similar  organisms  have  been  supposed  to  be  present  in  cancer. 

1  See  Lankester's  Treatise  en  Zoology,  Pt.  1,  First  Fascicle,  p.  37. 


MASTIGOPHORA  487 


Class  II. — Mastigophora 

These  are  protozoa  in  which  one  or  more  permanent  organs 
serving  for  locomotion  or  food  capture  are  present  in  the  form  of 
flagella.  As  a  rule  the  body  is  limited  by  either  a  cuticle  or  a 
differentiation  of  the  protoplasm  into  a  firmer  external  portion  or 
periplast.  One,  two,  or  more  flagella  may  be  present,  and  when 
multiple  are  arranged  in  various  ways.  Food-vacuoles  may  occur 
in  the  protoplasm,  also  contractile  vacuoles,  but  not  in  the  parasitic 
forms.  Various  other  granules,  including  chromatophores,  which 
generally  contain  chlorophyl,  may  be  present.  The  nuclear  appa- 
ratus is  usually  double,  consisting  of  a  large  principal  or  macro - 
nucleus,  and  a  small  or  micronucleus  or  blepharoplast  ;  the  latter 
is  not,  as  in  the  Infusoria,  composed  of  generative  chromatin,  and 
is  in  relation  with  the  locomotor  apparatus.  An  undulating  mem- 
brane, a  thin  protoplasmic  membrane  attached  to  one  aspect  of 
the  body  like  a  dorsal  fin,  may  be  present.  Euglena  is  a  common 
form  in  ditches,  and  Noctiluca  is  the  chief  cause  of  phosphorescence 
in  the  sea  ;  both  are  uniflagellate.  Volvox  and  Protococcus  are 
also  placed  by  some  in  this  group.  The  chief  parasitic  genera 
are : 

Trypanosoma  and  Trypanoplasma,  both  of  which  have  an  undu- 
lating membrane,  but  the  former  has  one  flagellum,  the  latter  two 
flagella,  one  at  each  end  of  the  body,  but  both  starting  from  the 
blepharoplast.  Spirochaeta  (see  p.  493). 

Herpetomonas,  like  Trypanosoma,  has  a  single  flagellum,  but  no 
undulating  membrane. 

Crithidia  has  a  pear-shaped  body  with  single  flagellum. 

Trichomonas,  also  somewhat  pear-shaped,  with  three  short 
flagella  and  an  undulating  membrane. 

The  trypanosomes  and  other  forms  living  in  the  blood  are  known 
as  haemoflagellates. 

Trypanosomata  * 

The  trypanosomes  are  all  parasitic  in  the  blood  of  vertebrates, 
and  a  blood-sucking  invertebrate  is  almost  invariably  concerned 
in  their  transmission.  In  the  case  of  each  pathogenic  trypanosome, 
some  indigenous  wild  animal,  tolerant  to  that  form,  serves  as  a 
reservoir  from  which  infection  is  derived. 

1  For  current  literature  on  Trypanosomes  and  trypanosome  diseases 
see  The  Tropical  Diseases  Bulletin. 


488  A  MANUAL  OF  BACTERIOLOGY 

A  trypanosome  has  a  slender,  flexible,  flattened  body,  one 
extremity  of  which  is  pointed,  the  other  passes  into  a  single  flagellum. 
A  delicate  undulating  membrane  passes  along  one  edge  of  the  body. 
The  organism  lives  in  the  plasma,  in  which  it  is  actively  motile,  the 
flagellated  end  being  usually  anterior,  and  measures  15-30  /*,  or 
even  40-50  /*,  in  length.  The  protoplasm  of  the  organism  is  finely 
granular,  and  near  the  centre  of  the  body  is  a  large  macronucleus, 
and  generally  between  it  and  the  non-flagellated  end  is  a  smaller 
micronucleus  or  blepharoplast.  From  the  latter  a  chromatin 
filament  starts,  runs  along  the  free  edge  of  the  undulating  membrane 
and  passes  into  the  flagellum.  Reproduction  takes  place  by  longi- 
tudinal division,  occasionally  probably  by  transverse  division,  and 
amoeboid  and  plasmodial  masses  may  be  found  in  the  internal 
organs  and  bone -marrow.  The  trypanosomes  have  great  mor- 
phological similarity,  which  renders  them  practically  indistinguish- 
able by  structural  characters.  They  can  usually  be  differentiated 
into  three  forms — indifferent,  male,  and  female — which  in  some 
cases  may  all  occur  together,  but  only  become  fully  differentiated 
in  an  invertebrate  host.  The  males  are  slender,  active,  only  slightly 
granular,  and  with  an  elongated  nucleus  ;  the  females  are  bulky, 
sluggish,  granular,  and  have  a  rounded  nucleus  ;  the  indifferent 
forms  are  intermediate.  The  males  usually  soon  die  off  unless  they 
conjugate  ;  the  indifferents  are  more  hardy,  the  females  most  so. 
The  sexual  forms  conjugate  in  an  invertebrate  host,  but  if  the  males 
have  died  off,  both  male  and  female  forms  may  be  reproduced  from 
the  females  by  a  process  of  parthenogenesis. 


Trypanosoma  Gambiense 

In  human  trypanosomiasis  and  sleeping-sickness  of 
West  and  Central  Africa,  a  trypanosome  Tr.  Gambiense 
is  the  causative  agent  (Plate  XXI.  a).  It  is  usually 
present,  though  scanty,  in  the  blood,  but  can  often  be 
found  in  numbers  in  the  fluid  aspirated  from  the  enlarged 
cervical  glands.  In  the  later  stages,  when  cerebral 
symptoms  ensue,  it  is  found  in  the  cerebro-spinal  fluid, 
but  scantily,  centrifuging  being  necessary  in  order  to 
demonstrate  the  parasites.  The  Tr.  Gambiense  is  patho- 
genic to  monkeys,  and  to  a  less  extent  to  white  rats  and 
guinea-pigs.  Cattle  and  certain  antelopes  and  other  wild 


TRYPANOSOME  GAMBIENSE  489 

game  may  act  as  reservoirs  of  the  parasite,  and  it  has 
been  seriously  suggested  to  kill  off  all  the  big  game  in 
the  affected  areas.  It  is  conveyed  by  a  tsetse-fly  (G. 
palpalis),  possibly  by  other  tsetses. 

The  tsetse  (and  possibly  other  biting  flies)  may  rarely  convey 
the  disease  by  direct  inoculation.  Generally  a  cycle  of  development 
is  passed  in  the  tsetse.  The  stages  of  this  are  not  known  with 
certainty,  but  Roubaud  has  observed  multiplication  of  the  parasites 
in  the  fly  and  the  development  of  Herpetomonas  forms.  According 
to  the  observations  of  Kleine  and  Bruce,  the  flies  become  infective 
about  thirty-four  days  after  feeding  and  remain  infective  for  at 
least  70-80  days,  and  probably  for  the  rest  of  their  lives. 

In  Rhodesia,  a  human  trypanosome  (Tr.  Khodesiense)  has  been 
found  which  is  probably  distinct  from  Tr.  gambiense,  and  the 
O.  palpalis  does  not  occur  in  the  district.  The  macronucleus  of  the 
parasite  is  situated  between  the  blepharoplast  and  the  posterior  end. 

In  Brazil  another  human  trypanosome-like  parasite  has  been 
discovered  by  Chagas  (Tr.  or  Schizotrypanum  cruzi),  which  is 
conveyed  by  a  bug  (Conorhinus  megistus). 

Tr.  Brucei  is  the  causative  parasite  of  nagana  or  tsetse- 
fly  disease  of  horses  in  Africa. 

Nagana  is  met  with  in  large  tracts  of  country  in  Zululand 
and  West  Africa.  It  especially  attacks  the  equines — 
horse,  mule,  and  ass — in  which  it  is  very  fatal.  The 
animals  become  anaemic  and  emaciated,  there  is  a  discharge 
from  the  eyes  and  nose,  staring  coat,  swelling  of  the  legs 
and  neck,  and  fever.  The  animal  dies  two  to  six  weeks 
after  infection.  Oxen  are  also  attacked,  but  a  small 
proportion  recover.  The  dog,  cat,  rabbit,  guinea-pig, 
mouse,  and  rat  may  be  infected  by  inoculation  with  the 
fresh  blood  of  a  diseased  animal.  In  infected  animals 
the  trypanosome  is  generally  abundant  in  the  blood  and 
spleen.  The  Tr.  Brucei  can  be  cultivated,  though  with 
difficulty,  on  rabbit-blood  agar — melted  sterile  agar  cooled 
to  45°  C.  -f  sterile  defibrinated  rabbit's  blood  warmed  to 
45°  C.,  mixed  and  allowed  to  solidify  in  the  sloping  position 
(Novy  and  McNeal).  The  disease  is  conveyed  through  the 


490  A  MANUAL  OF  BACTERIOLOGY 

bites  of  a  tsetse-fly  (Glossina  morsitans).  The  trypanosome 
is  believed  to  live  in  the  big  game,  from  whence  it  is  trans- 
mitted to  horses  entering  the  infected  localities.  The 
blood  loses  its  infective  properties  usually  within  twenty- 
four  hours  after  being  withdrawn. 

Surra  attacks  horses  in  Burma,  Mauritius,  and  the 
Philippines,  and  is  pathogenic  to  the  same  animals  as 
nagana,  and  in  the  blood  a  parasite  (Tr.  Evansi)  similar 
to  that  in  nagana.  but  more  active,  was  observed  by 
Evans.  Surra  is  probably  spread  by  certain  biting  flies 
belonging  to  the  Tabanidce. 

The  tsetse  flies  (Glossina)  belong  to  the  house-fly  order  (Muscidse) 
and  have  a  general  resemblance  to  a  house-fly,  but  when  at  rest 
the  wings  fold  completely  over  each  other.  The  proboscis  is  long 
and  straight  and  the  wing  venation  is  characteristic,  especially  the 
fourth  longitudinal  vein,  which  makes  two  bends.  Instead  of  laying 
eggs,  the  female  extrudes  a  single  full-grown  larva.  They  are 
confined  to  Africa  and  Arabia  ;  some  sixteen  species  have  been 
differentiated,  and  they  occur  in  the  vicinity  of  water  on  the  edge 
of  forest  land  ("  fly-belts  "). 

Tr.  equinum  attacks  horses  in  South  America,  causing  weakness 
and  paresis  of  the  hindquarters  ("ma/  de  caderas").  Cattle  are 
immune,  most  other  animals  susceptible. 

Tr.  Theileri,  the  largest  trypanosome  known  (50-60  p  in  length), 
is  found  in  cattle  in  ISouth  Africa,  and  is  not  pathogenic  to  any 
other  animal. 

Tr.  dimorphum  occurs  in  two  forms,  large  and  small,  in  horses 
in  Africa.  Is  pathogenic  to  most  animals. 

Dourine,  a  venereal  disease  of  the  horse  met  with  in  North  Africa, 
Spain,  and  Hungary,  is  due  to  the  Tr.  equiperdum,  which  is  conveyed 
by  direct  contact,  and  is  mainly  confined  to  the  lesions,  being 
scanty  in  the  blood.  It  is  pathogenic  to  the  ordinary  laboratory 
animals. 

In  rats  a  non-pathogenic  trypanosome  was  found  by  Lewis 
(Tr.  Lewisi).  It  is  especially  met  with  in  sewer-rats,  but  also 
occurs  in  field-rats  (Crookshank).  It  is  somewhat  shorter  and 
thinner  than  the  Tr.  Brucei,  and  there  are  other  small  differences 
between  the  two  forms.  With  the  exception  of  rats  and  mice, 
and  to  a  less  Extent  guinea-pigs,  other  animals  cannot  be  infected 
with  the  Tr.  Lewisi.  It  may  be  kept  alive  for  long  periods  in  the 


LEISHMANIOSIS  491 

blood  placed  in  a  refrigerator,  whereas  the  Tr.  Brucei  soon  dies 
under  the  same  conditions.  The  two  forms  do  not  protect  against 
each  other.  The  Tr.  Lewisi  is  readily  cultivated  on  rabbit-blood 
agar  and  is  transmitted  by  the  rat-flea,  in  which  it  seems  to  pene- 
trate into  the  epithelial  cells  of  the  gut  and  there  undergoes  a  process 
of  multiplication.1  It  is  passed  in  the  faeces  of  the  flea  and  a  rat 
ingesting  the  infected  faeces  becomes  infected. 

A  number  of  other  trypanosomes  have  been  found  in  the  lower 
animals,  birds,  fish,  reptiles,  and  amphibians.  A  large  and  charac- 
teristic one  is  generally  present  in  the  blood  of  the  eel. 

The  trypanosomes  are  usually  agglutinated  when  mixed  with 
the  serum  from  an  infected  animal. 

Hewlett  was  unable  to  obtain  any  toxic  or  immunising  substance 
from  ground-up  trypanosomes  (Tr.  Brucei).2 

Levaditi  and  Twort  3  have  found  that  the  filtrate  of  broth  cultures 
of  B.  subtilis  is  markedly  trypanocidal  in  vitro  but  not  in  vivo. 


Examination  of  Trypanosomes,  etc. 

The  trypanosomes,  if  numerous,  are  readily  observed  in  the 
fresh  blood.  A  very  shallow  cell  may  be  formed  on  a  slide  by 
ringing  with  melted  paraffin.  For  stained  preparations  theLeish- 
man  stain  (see  "  Malaria  ")  or  the  Heidenhain  method  (p.  485) 
may  be  employed.4 

Leishmaniosis 

This  term  is  applied  to  a  group  of  diseases,  caused  by 
a  similar  parasite,  and  widely  distributed  in  tropical 
and  sub-tropical  countries  of  the  old  and  new  world.5 

In  kala-azar  or  tropical  splenomegaly,  a  disease  met 
with  in  India,  Assam  and  the  East,  a  small  parasite,  the 
Leishman-Donovan  body,  occurs  in  large  numbers  in  the 
spleen  and  liver,  also  in  the  lymphatic  glands,  lungs,  and 
intestinal  submucosa,  and  in  large  rnononuclear  leucocytes 

1  Minchin  and  Thompson,  Brit.  Med.  Journ.,  1911,  vol.  ii,  p.  361. 

2  Proc.  Roy.  Soc.  Lond.,  B.,  vol.  Ixxxiv,  1911,  p.  56. 

3  Ccmp.  Rend.  Soc.  Biol,  vols.  Ixx  and  Ixxi,  1911. 

4  For  a  special  method  of    staining,  see  Plimmer,  Proc.  Roy.  $oc.. 
Lond.,  B.  vol.  Ixxix,  1907,  p.  102. 

5  See  Hewlett,  Practitioner,  1911,  July,  p.  109. 


492  A  MANUAL  OF  BACTERIOLOGY 

and  endothelial  cells.  The  bodies  are  small  (2-3  //),  round, 
ovoid,  or  oat-shaped  masses  of  protoplasm,  apparently 
encapsuled,  and  contain  two  chromatin  masses,  one  large 
and  oval,  staining  pale  red  with  Leishman's  stain,  the 
other  small  and  rod-shaped,  and  staining  deep  red  with 
Leishman  (Fig.  56,  a).  They  sometimes  occur  in  masses 
(Fig.  56,  c).  Leishman  considered  the  bodies  to  be  degene- 
rate trypanosomes,  but  the  organism  is  now  considered 


FIG.  56. — a.  The  Leishman-Donovan  body.  b.  The  flagellated 
form  developing  in  citrated  blood,  c.  Seven  parasites  in  a 
farge  mononuclear  leucocyte.  (After  James.  Patton,  and 
Rogers.) 

to  belong  to  a  distinct  genus,  and  is  termed  Leishmania 
Donovani.  Rogers  succeeded  in  cultivating  it  in  citrated 
blood  at  20°-25°  C.,  in  which  it  develops  into  a  flagellated 
form  like  Herpetomonas  (Fig.  56,  6).1  The  parasite  is  not 
inoculable  into  animals,  and  it  is  probably  transmitted 
to  man  by  a  bug  (?  a  Conorhinus). 

The  bodies  are  well  shown  in  smears  stained  with  the 
Leishman  stain. 

In  Oriental  sore,  or  Delhi  boil,  a  parasite  practically 
identical  with  the  Leishman-Donovan  body  is  present, 
but  as  the  two  diseases  run  a  totally  different  course,  it  is 

1   Brit.  Med.  Journ.,  1907,  vol.  i,  p.  427  et  seg. 


SPIROCHAETOSIS  493 

probably  a  distinct  species  (L.  tropica).  On  cultivation 
it  develops  a  flagellated  form.  The  disease  has  a  seasonal 
prevalence,  and  Wenyon  suggests  that  it  is  conveyed  by 
a  mosquito,  a  species  of  Stegomyia. 

In  N.  Africa  Nicolle  has  observed  a  Leishmaniosis  of 
children  due  to  another  species  (L.  infantum).  It  is  trans- 
missible to  the  dog  and  monkey,  and  can  be  cultivated. 
The  disease  has  recently  been  found  all  along  the  Mediter- 
ranean littoral. 

Spirochaetosis l 

Diseases  caused  by  infection  with  spirochaetes. — The  spirochaetae 
are  delicate,  undulating,  or  somewhat  spirillar,  filiform  parasites 
occurring  in  the  blood  of  man,  mammals,  birds,  shell-fish,  etc.  The 
filaments  taper  to  a  point  at  the  ends,  are  flexible  and  motile, 
coiling  and  uncoiling,  are  described  as  having  two  nuclear  masses, 
and  some  possess  an  undulating  membrane,  like  trypanosomes, 
but  in  the  smaller  forms  no  definite  structure  can  be  made  out. 
They  are  now  generally  regarded  as  protozoa,  but  some  still  con- 
sider them  to  be  bacteria.  Bacterial  cells  are  never  pointed,  nor 
do  they  show  the  coiling  movements  of  spirochaetes  ;  motility  is 
produced  by  flagella,  which  are  absent  from  most  spirochaetes 
(statements  to  the  contrary  are  due  to  errors  of  observation  and 
technique),  and  periodicity  is  not  exhibited  by  bacteria.  Spiro- 
chaetes are  said  to  multiply  by  longitudinal  fission,  while  fission 
in  bacteria  is  transverse  (Dobell  states  that  multiplication  is  always 
by  transverse,  but  multiple,  fission.  See  p.  12)  ;  they  react  in 
some  cases  to  drugs  (e.g.  salvarsan)  like  trypanosomes,  are  much 
more  sensitive  to  the  action  of  immune  sera  than  bacteria  are,  and 
are  transmitted  by  insects.  Noguchi  has  cultivated  certain  spiro- 
chaetes of  the  mouth  and  relapsing  fever  by  a  method  similar  to 
that  which  he  employed  for  syphilis  (p.  497).  For  the  saprophytic 
spirochaetes  a  small  quantity  of  oxygen  is  required,  for  the  blood 
spirochaetes  absolute  anaerobiosis  is  necessary  as  in  the  case  of 
syphilis. 

Schaudinn  believed  that  many  so-called  spirochaetes  may  be 
connected  with  the  trypanosomes.  In  8.  plicatilis  he  described 
the  presence  of  a  thread-like  nucleus  and  of  chromidia,  and  of 
an  undulating  membrane,  but  flagella  are  absent.  In  the  little 

1  See  Nuttall,  Jaurn.  Roy.  Inst.  Pub.  Health,  xvi,  1908,  p.  449. 


494  A  MANUAL  OF  BACTERIOLOGY 

owl  minute  slender  trypanosomes  occur  ;  these  later  penetrate 
leucocytes,  and  develop  into  relatively  very  large  trypanosome 
forms  (which  have  been  termed  Leucocytozoa).  These  intra- 
oorpuscular  forms  are  male  and  female  gametocytes,  the  male  being 
smaller  and  more  slender  than  the  female.  If  taken  into  the  gnat's 
stomach,  the  male  gametocytes  give  rise  to  eight  microgametes  by 
a  process  of  sporulation,  which  fertilise  the  macrogamete,  and  the 
resulting  zygote  ultimately  forms  by  sporulation  an  immense 
number  of  spirochaetes. 

In  the  case  of  a  Halteridium  parasite  of  the  little  owl  (Athene 
noctua),  Schaudinn  claimed  to  have  shown  that  it  is  a  stage  of  a 
trypanosome  (jP.  noctuce)  which  is  disseminated  by  the  common  gnat. 
His  observations  have  not  been  confirmed,  and  Novy  and  McNeal 
believe  that  Schaudinn  was  dealing  with  a  double  infection  of  both 
a  trypanosome  and  a  Halteridium,  not  that  one  was  transformed 
into  the  other. 

Spirochaeta  recurrentis  (Obermeieri). — Found  in  the 
blood-plasma,  not  in  the  corpuscles,  in  relapsing  fever 
during  the  febrile  paroxysms.  It  is  very  slender  and 
delicate,  measuring  12  to  16  //.  in  length,  and  actively 
motile  (Plate  XXI.  6).  Bugs  were  formerly  supposed 
to  transmit  this  parasite,  but  Nicolle,  Blaizot  and  Conseil 
have  established  the  body  louse  as  the  agent  of  trans- 
mission. Infection  is  however  not  due  to  the  bite  of  the 
louse,  but  to  lice  being  crushed  by  the  victim's  scratching 
and  the  contents  of  the  lice  rubbed  into  the  abrasions. 
The  lice  not  only  retain  the  infection  for  the  rest  of  their 
lives,  but  the  spirochaetes  pass  into  their  eggs,  and  these 
eggs  and  the  larvae  hatched  from  them  may  similarly  be 
infective  to  man.  It  is  inoculable  into  monkeys,  and, 
less  readily,  into  rats. 

Noguchi  and  Hata l  have  cultivated  this  form  :  the 
latter  in  a  medium  consisting  of  one  part  of  horse- serum 
and  two  parts  of  saline.  This  mixture  is  placed  in  tubes 
to  a  depth  of  7  cm.,  which  are  then  heated  slowly  in  a 
water-bath  from  58°  C.  to  70°  C.,  at  which  they  are  kept 

1  Centr.f.'Bakt.,  Abt.  I  (Originate),  vol.  Ixxii,  1913,  p.  107. 


I 

PLATE  XXI. 


a.  Trypanosoma  Gambiense.     Smear  of  blood  of  inoculated  rat. 
X  1500. 


6.  Spirochaeta  recurrentis  (Obermeieri).     Smear  of  blood. 
X  1500. 


SPIEOCHAETA  PERTENUIS  495 

for  thirty  minutes.  Small  pieces  of  rabbit  kidney  are  then 
pushed  to  the  bottom  of  the  tubes  and  the  incubation 
must  be  carried  out  anaerobically. 

It  is  probable  that  the  spirachaetes  of  relapsing  fever 
in  different  countries  are  distinct  species. 

Spirochaeta  Duttoni. — Found  in  the  blood-plasma  in 
African  relapsing,  or  tick,  fever.  It  closely  resembles 
the  S.  recurrentis,  but  is  more  readily  inoculable  into 
rats,  mice,  and  guinea-pigs,  and  the  one  does  not  protect 
against  the  other.  It  is  conveyed  by  a  tick,  Ornithodoros 
moubata,  the  malpighian  secretion  of  which  is  the  principal 
infective  agent.  The  eggs  of  infected  ticks  are  also  in- 
fected, and  the  infection  may  be  transmitted  to  the  third 
generation  of  ticks. 

Duval  and  Todd  l  state  that  multiplication  of  S.  Duttoni 
takes  place  in  vitro  in  a  culture  medium  made  with  hens' 
eggs  and  mouse  blood.  Leishman  believes  that  certain 
chromatin  bodies  present  in  the  eggs  and  nymphs  of  the 
ticks  are  the  developmental  forms  of  the  spirochaetes. 

Blood  spirochaetes  have  been  found  in  many  animals, 
e.g.  cattle  (S.  Theileri),  mice  (S.  muris),  fowls  (S.  galli- 
narum),  and  geese  (S.  anserina). 

Spirochaeta  pertenuis. — Castellani 2  found  in  the 
yaws  (frambcesia)  granulomata  a  delicate  spirochaete 
resembling  the  S.  pallida  of  syphilis  closely,  but  even 
more  delicate  and  difficult  to  stain  than  the  latter  organism, 
and  named  the  S.  pertenuis.  It  is  present  also  in  the 
spleen  and  lymphatic  glands  in  the  disease  and  in  inoculated 
monkeys.  Rabbits  can  be  inoculated  in  the  testicle  and 
Noguchi  has  obtained  cultures. 

Some  observers  have  supposed  yaws  to  be  a  manifesta- 
tion of  syphilis,  but  (1)  syphilitic  patients  can  be  inoculated 
with  yaws ;  (2)  syphilis  may  supervene  on  yaws ; 

1  Lancet,  1909,  vol.  i,  p.  834. 

2  Brit.  Med.  Journ.,  1907,  vol.  ii,  p.  1511. 


496  A  MANUAL  OF  BACTERIOLOGY 

(3)  Neisser  and  Castellani  have  shown  that  monkeys 
inoculated  with  syphilis  are  not  immune  to  yaws,  and  vice 
versa  ;  and  (4)  Castellani 1  has  shown  that  the  yaws  antigen 
and  anti-bodies  are  distinct  from  the  syphilis  antigen  and 
anti-bodies,  though  the  ordinary  Wassermann  test  may 
react  with  yaws. 

Spirochaetes  are  also  present  in  the  ulcerating  granuloma 
of  the  pudenda  of  Guiana  (Wise)  and  Australia,  in  malig- 
nant growths,  in  ulcers,  in  the  mouth  (p.  570),  and  in 
Vincent's  angina  (p.  296). 

Staining  methods. — Blood-smears  may  be  stained  with 
the  Leishman  or  Giemsa  stain  (p.  102). 

Trichomonas  vaginalis. — This  parasite  is  found  in  the  acid  vaginal 
mucus  in  50  per  cent,  of  those  examined.  It  must  not  be  mistaken 
for  a  spermatozoon.  It  is  a  pear-shaped  body,  measuring  12  to 
30  p.  in  length,  and  from  the  blunt  end  three  flagella  are  given  off. 

A  much  smaller  species,  T.  intestinalis,  measuring  4  to  15  p.,  has 
been  met  with  in  the  intestinal  canal  of  man  in  conditions  associated 
with  diarrhoea. 

Syphilis 

Various  bacterial  organisms  have  been  described  in 
this  disease,  e.g.  by  Lustgarten,  Eve  and  Lingard,  Van 
Niessen,  de  Lisle  and  Jullien,  etc.,  and  bodies  regarded 
as  protozoa  by  Siegel,  de  Korte,  and  others.  In  March 
1905,  Schaudinn  2  noted  the  constant  presence  of  a  spiriform 
organism  or  spirochaeta  (S.  pallida,  or  Treponema  or 
Spironema  pallidum)  in  various  lesions  in  acquired  and 
congenital  syphilis.  The  T.  pallidum  varies  from  6  to  15  /m 
in  length,  averaging  8-9  /UL  (Plate  XXII.  a  and  6).  It  is 
much  more  attenuated  than  the  majority  of  spirochaetes, 
having  a  maximum  thickness  of  0'3  yu,  has  from  three  to 
twelve,  usually  from  six  to  eight,  twists,  forming  a  close, 

1  Journ.  of  Hygiene,  vol.  vii,  1907,  p.  558. 

2  Arbeit,  a.  d.  Kaiser.  Gesurtdheitsamte,  xx.  1905. 


PLATE  XXII. 


a.  Treponema  pallidum  from  condyloma  (T.  pallidum  with 
Spirochaeta  refringens).     Indian-ink  method.      X  1000. 


b.  Treponema  pallidum.     Smear  from  condyloma.     Giemsa. 
X  1500. 


SYPHILIS  497 

regular,  and  narrow  spiral,  is  actively  motile,  possessing 
a  single  delicate  flagellum  at  each  end,  and  it  may  have 
an  undulating  membrane.  It  stains  feebly  and  with 
difficulty.  Another  spirochaete,  the  S.  refringens,  fre- 
quently accompanies,  and  must  not  be  mistaken  for,  the 
T.  pallidum  in  ulcerating  lesions  ;  the  former  is  more 
refractile  and  coarser,  has  fewer  twists  and  forms  a  wider 
spiral,  and  stains  deeper  and  more  readily  than  the  latter. 
The  T.  pallidum  is  found  generally  in  all  primary  and 
secondary  lesions  of  syphilis,  e.g.  the  primary  sore  and 
adjacent  lymphatic  glands,  in  the  papular  and  roseolar 
eruptions,  in  condylomata  and  mucous  patches.  It  has 
also  occasionally  been  found  in  the  spleen  and  blood.  In 
congenital  syphilis  the  T.  pallidum  is  met  with  in  the 
bullous  eruptions,  blood,  and  organs,  and  is  particularly 
abundant  in  the  spleen  and  liver  (Plate  XXIII.  a). 

Tertiary  lesions  are  generally  considered  to  be  non- 
infective,  and  the  T.  pallidum  is  usually  difficult  to  find 
in  them.  It  has,  however,  been  detected  in  the  peripheral 
portions  of  gummata  and  in  syphilitic  aortitis,  and  may 
persist  in  the  body  for  years  after  the  primary  lesion. 
Noguchi,  after  a  careful  search,  has  detected  the  spiro- 
chaete in  the  brain  in  cases  of  general  paralysis  (in  48 
cases  out  of  200  examined)  and  also  in  the  posterior  columns 
in  a  case  of  tabes. 

The  T.  pallidum  is  now  universally  regarded  as  the 
specific  organism  of  syphilis,  being  present  not  only  in 
the  human  lesions  but  in  experimental  lesions  of  inoculated 
apes  (see  below).  It  must  be  recognised  that  spirochaetes 
are  of  frequent  occurrence  in  various  non-syphilitic  ulcera- 
ting and  other  lesions,  e.g.  in  the  mouth  and  in  pyorrhoea, 
in  yaws  and  ulcerating  granuloma  (in  yaws  they  are  specific 
forms,  see  p.  495),  in  ordinary  ulcers  and  in  carcinomatous 
tumours.  Generally  the  T.  pallidum  can  be  distinguished 
microscopically  from  the  other  species,  but  care  is  necessary. 

32 


498  A  MANUAL  OF  BACTERIOLOGY 

When  material  from  a  rhesus  monkey  inoculated  with 
syphilis  is  placed  in  collodion  sacs  which  are  introduced 
into  the  peritoneal  cavity  of  another  monkey,  a  great 
multiplication  of  the  organism  takes  place  in  the 
contents  of  the  sacs  a  month  after  the  operation.1 
Noguchi  has  obtained  cultures  of  the  Treponema  pallidum 
by  making  use  of  serum  water  (serum  1  part,  water  3 
parts),  sterilised  for  fifteen  minutes  at  100°  C.  on  three 
days,  to  which  fragments  of  fresh  sterile  tissue  of  a  rabbit 
(kidney,  heart-muscle)  were  added.  Rabbits  are  inoculated 
with  syphilis  in  the  testicle  and  the  spirochaete- containing 
testicular  material  is  employed  to  inoculate  the  tubes, 
which  are  then  incubated  at  35°-37°  C.  under  strictly 
anaerobic  conditions.  Multiplication  of  the  spirochaetes 
commences  forty- eight  hours  after  inoculation.  The 
primary  cultures  are  somewhat  difficult  to  obtain,  but 
once  obtained  sub-cultivation  is  easy.  Both  thick  and 
thin  forms  of  the  Treponema  were  obtained,  which  Noguchi 
considers  may  be  distinct  varieties. 

Metchnikoff  and  Roux  (also  Griinbaum)  found  that  the 
chimpanzee  is  very  susceptible  to  syphilis,  and  can  readily 
be  inoculated  from  manj  the  T.  pallidum  being  found  in 
the  lesions. 

Macacus  rhesus  is  also  somewhat  susceptible,  likewise 
the  M.  cynomolgus  and  the  Chinese  bonnet  monkey,  but 
not  the  mandril.  By  several  passages  through  a  rhesus 
monkey  the  syphilitic  virus  becomes  attenuated,  so  that 
in  man  it  produces  merely  a  local  lesion.2  Syphilis  may 
also  be  inoculated  on  the  eye  or  testicle  of  the  rabbit. 

Although  the  central  nervous  systems  of  rabbits  and 
monkeys  are  refractory  to  direct  inoculation  with  T. 
pallidum,  Noguchi  has  succeeded  in  inducing  some  of  the 
symptoms  (convulsions)  and  lesions  of  general  paralysis 

1  Levaditi  and  Mclntosh,  Ann.  de  Vlnst.  Pasteur,  xxi,  1907. 

2  Metchnikoff,  Journ.  of  Prev.  Med.,  1906,  August. 


PLATE  XXIII. 


a.  Treponcma  pallidum.     Section  of  liver  of  fetus  (congenital 
syphilis.)     Levaditi's  method.      X  1500. 


b,  Coccidium  oviforme.     Section  of  rabbit's  liver,      x  350. 


SYPHILIS  499 

in  these  animals  by  the  following  method.  Intravenous 
inoculations  of  dead  spirochaete  cultures  were  given  every 
five  days  over  a  period  of  five  months,  an  interval  of  five 
months  was  then  allowed  to  elapse,  and  finally  the  living 
spirochaetes  were  introduced  into  the  brain,  subdurally 
or  intra-cerebrally. 

Attempts  by  Metchnikoff  and  Roux  to  prepare  an  anti- 
syphilitic  serum  by  inoculating  apes  and  goats  with 
syphilitic  virus  proved  unsuccessful  (as  did  earlier  experi- 
ments with  other  animals  by  Hericourt  and  Richet).  The 
syphilitic  virus  as  ordinarily  introduced  into  man  by  sexual 
intercourse  probably  takes  some  hours  to  become  gene- 
ralised, for  Metchnikoff  found  experimentally  in  apes  that 
if  the  seat  of  inoculation  were  treated  with  a  calomel 
ointment  up  to  eighteen  hours  after  inoculation  infection 
was  prevented. 

By  triturating  cultures  of  the  Treponema  in  salt-solution, 
heating  to  60°  C.  for  sixty  minutes,  and  adding  O5  per  cent, 
of  carbolic  acid,  Noguchi  has  prepared  an  agent,  termed 
Luetin,  which  can  be  used  for  a  cutaneous  reaction  for 
the  diagnosis  of  syphilis.  In  syphilitic  infection  redness, 
sometimes  becoming  pustular,  develops  at  the  site  of 
inoculation. 

The  syphilitic  virus  does  not  pass  through  aBerkefeld 
filter,  and  hence  is  not  ultra-microscopic.  It  is  readily 
destroyed  by  heat  (52°  C.)  and  antiseptics.  Treatment 
with  mercury  and  with  salvarsan  ("  606 ")  and  neo- 
salvarsan  cause  diminution  or  disappearance  of  the  spiro- 
chaetes. 

In  central  nerve  lesions  salvarsan  is  more  effective 
when  injected  into  the  central  nervous  system,  but  this 
procedure  is  not  free  from  danger.  To  obviate  this,  the 
salvarsan  may  be  injected  intravenously  and  then  some 
of  the  patient's  serum  is  injected  into  the  spinal  canal  by 
lumbar  puncture. 


500  A  MANUAL  OF  BACTERIOLOGY 


Examination  for  the  T.  pallidum 

1.  Examination  in  fresh  preparations. — Scrapings  from  the  deeper 
layers  of  the  chancre,  etc.,  may  be  emulsified  in  physiological  salt 
solution  and  examined  microscopically,  particularly  with  dark- 
ground  illumination  (p.  139). 

Another  useful  method  is  the  Indian-ink  method.  A  scraping  is 
obtained  from  the  lesion  as  above,  and  the  fluid  thus  obtained  is 
placed  on  a  slide  and  an  equal  quantity  of  ink  added.  The  ordinary 
commercial  Indian  inks  may  be  used,  Giinther  Wagner's  being 
particularly  good  (p.  81).  The  ink  must  be  examined  microscopi- 
cally to  prove  the  absence  of  spirillar  forms,  which  sometimes  occur 
in  it.  The  serum  and  the  ink  are  then  rapidly  and  thoroughly 
mixed  and  smeared  over  the  slide  so  that  a  pale  brown  colour 
results.  The  material  dries  in  a  minute  or  slightly  less,  and  may 
be  examined  directly  with  the  oil-immersion  lens,  or  the  wet  pre- 
paration may  be  covered  with  a  cover-glass  and  examined. 

The  preparations,  which  keep  for  a  considerable  time,  show  the 
red  blood-cells  as  large  clear  circular  areas  in  a  brownish -black 
field,  the  bacteria  and  debris  as  white  rods,  dots,  etc.,  and  spiro- 
chaetes,  as  clear  white  spirals  (Plate  XXII.  a). 

It  is  particularly  important  in  using  this  method  that  in  so  far 
as  possible  serum  alone  be  used,  and  that  a  minimal  amount  of 
mucous  material  or  fibrin  be  mixed  with  the  ink.  The  presence  of 
mucus  results  in  the  taking  up  of  a  large  amount  of  the  colouring 
matter  of  the  ink,  with  the  result  that  a  smear  of  the  requisite 
colour  and  thickness  cannot  be  made.  If  too  much  serum  is  used 
the  albuminous  material  appears  to  precipitate  the  colour  from  the 
fluid  and  a  finely  granular  appearance  is  seen  microscopically,  which 
is  practically  worthless  for  diagnostic  purposes.  Again,  if  too 
much  ink  is  used,  the  surface  of  the  smear  is  increased  in  size  to 
such  an  extent  that  the  task  of  examining  it  thoroughly  is  greatly 
lengthened. 

Coles  *  notes  a  useful  point  in  the  recognition  of  the  treponema, 
namely,  that  if  the  number  of  turns  of  the  spiral  of  the  syphilitic 
spirochaete  be  counted,  six  or  seven  turns  will  be  found  in  a  length 
equal  to  the  diameter  of  a  red  blood-cell.  The  distance  from  the 
top  of  one  spiral  to  the  next  is  from  1  to  1-2  p.  As  red  blood-cells 
measure  about  7-5  p  in  diameter,  on  an  average  six  or  seven  turns 
will  be  equal  to  the  diameter  of  a  red  blood-cell.  The  treponema 
varies  in  length  from  6  to  15  p.,  or  even  more,  and  consequently 
1  Brit.  Med.  Journ.,  May  8,  1909. 


THE  WASSERMANN  REACTION  501 

contains  from  six  to  fourteen  and  sometimes  twenty  or  more  turns. 
This  measurement  of  the  length  of  the  spiral  is  usually  possible, 
and  is  of  the  greatest  value  in  identifying  the  treponema. 

2.  Stained  preparations. — Smears  from  chancres,  etc.,   may  be 
stained  by  the  Giemsa  method. 

The  smears  are  fixed  for  ten  minutes  in  absolute  alcohol.  The 
preparations  are  then  stained  in  a  dilute  solution  of  the  Giemsa 
solution  for  two  to  twenty-four  hours,  washed  in  distilled  water, 
dried,  and  mounted.  (The  dilute  Giemsa  is  prepared  by  adding 
one  drop  of  the  Giemsa  stain  to  a  cubic  centimetre  of  distilled  water, 
and  rendering  alkaline  with  one  drop  of  0-01  per  cent,  potassium 
carbonate  solution.)  The  preparations  may  also  be  stained  in  the 
undiluted  Giemsa  stain  for  half  to  six  hours.  Leishman's  solution 
may  also  be  used  or  the  Giemsa  method  described  under  "  Malaria." 

Sections  may  be  stained  by  Levaditi's  method  : 

(1)  Fix  pieces  of  tissue  about  1  mm.  thick  in  10  per  cent,  formalin 
for  twenty -four  hours. 

(2)  Wash  in  water,  and  harden  in  96  per  cent,  alcohol  for  twenty- 
four  hours. 

(3)  Wash  in  distilled  water  for  some  minutes  (until  pieces  sink). 

(4)  Place  in  3  per  cent,  silver  nitrate  solution  at  37°  C.  for  three 
to  five  days  in  the  dark. 

(5)  Wash  in  distilled  water  for  some  minutes,  and  then  place 
in  the  following  solution  at  room  temperature  for  twenty-four  to 
forty-eight  hours. 

Pyrogallic  acid    ......     2-4  grm. 

Formalin    .......         5  c.c. 

Distilled  water    .          .  .          .          .     100  c.c. 

(6)  Wash  in  distilled  water,  dehydrate  in  absolute  alcohol,  clear 
in  xylol,  embed  in  paraffin,  cut,  and  mount. 

The  spirochaetes  are  stained  black  or  brown  (Plate  XXIII.  a), 
the  tissues  yellow. 

Some  have  asserted  that  the  spirochaetes  seen  in  the  tissues  after 
staining  by  this  method  are  artifacts  or  are  composed  of  filaments 
of  elastic  tissue.1 

3.  The  Wassermann  reaction  or  antigen  test. — This  is  applied  in 
the  diagnosis  of  syphilitic  conditions,  and  as  a  confirmatory  test 
of  the  syphilitic  nature  of  such  conditions  as  tabes  dorsalis  and 
general  paralysis  of  the  insane.     The  test  is  based  on  complement- 
fixation  (p.    183).     In  this  method  an   "  antigen  "   consisting  of 
micro-organisms,  or  an  extract  thereof,  fixes  its  homologous  immune- 

1  See  Saling  and  Miihlens,  Centr.  f.  Bakt.  (Orig.),  xlii  and  xliii. 


502  A  MANUAL  OF  BACTERIOLOGY 

body,  and  the  complex  then  takes  up  complement  ;  this  is  demon- 
strated by  the  use  of  a  haemolytic  system  (p.  184). 

As  a  matter  of  fact,  however,  the  Wassermann  reaction,  as  it 
is  preferably  termed,  is  apparently  not  a  true  antigen  reaction,  for 
substances  may  be  used  as  antigen  which  are  soluble  in  alcohol, 
and  various  non-specific  bodies  may  be  similarly  employed.  More- 
over, the  nature  of  the  substances  which  act  as  amboceptor  and 
fix  the  complement  is  uncertain  ;  some  regard  them  as  globulins, 
others  as  lipoids,  and  while  Wassermann  considered  them  to  be 
specific  anti-bodies,  others  believe  them  to  be  derived  from  a  peculiar 
degeneration  or  breaking  down  of  the  tissues  in  syphilis.  Again, 
the  reaction  is  not  confined  to  syphilis  :  it  may  also  be  obtained 
with  the  syphilitic  "  antigen  "  in  malaria,  trypanosomiasis,  yaws, 
leprosy,  and  the  early  stage  of  scarlatina. 

In  the  original  method  a  fresh  salt-solution  extract  of  the  liver 
of  a  syphilitic  fetus  was  used  as  the  "  antigen."  Levaditi  employed 
a  similar  extract  of  the  dried  and  powdered  liver.  The  test-sub- 
stance was  the  blood -serum  inactivated  by  heating  to  56°  C.  for 
half  an  hour  or  cerebro -spinal  fluid  of  the  patient.  The  complement 
was  guinea-pig  serum,  and  the  haemolytic  system  sheep's  corpuscles, 
and  a  serum  haemolytic  for  these  corpuscles. 

Many  modifications  of  this  method  have  since  been  introduced 
both  as  regards  the  reagents  employed — antigen,  complement,  and 
haemolytic  system — and  the  manner  of  carrying  out  the  test.  These 
may  now  be  briefly  considered  and  the  manner  of  carrying  out  the 
test  described. 

(a)  Antigen.  The  various  substances  which  have  been  used  as 
antigen  include  : 

1.  A  watery  or  alcoholic  extract  of  syphilitic  fetal  liver. 

2.  Alcoholic  extract  of  normal  liver  or  heart-muscle  1(human, 

ox,  sheep  or  guinea-pig),  with  or  without  previous  extrac- 
tion with  acetone. 

3.  Alcoholic  extract  of  normal  heart-muscle  with  the  addition 

of  cholesterin. 

4.  Various   artificial   mixtures,    e.g.   lecithin   and   cholesterin, 

sodium  glycocholate  or  taurocholate. 

5.  Extracts  of  pure  cultures  of  the  Treponema  pallidum  obtained 

by  Noguchi's  method. 

Probably  the  most  widely  employed  antigen  at  the  present  day 
is  number  3,  the  so-called  "  Sachs  antigen." 

1  Heart  muscle  is  peculiar  in  that  it  contains  a  large  amount  of  lipoid 
substances. 


THE  WASSERMANN  REACTION  503 

• 

This  is  prepared  by  extracting  1  grm.  of  heart-muscle  with  10  c.c 
of  absolute  alcohol  for  three  or  four  days.  To  4  parts  of  this 
extract  add  5  parts  of  a  1  per  cent,  alcoholic  solution  of 
cholesterin. 

The  solution  of  antigen  must  be  tested  as  regards  its  possible  com- 
plement fixing  properties  alone  and  in  the  presence  of  a  known 
positive  syphilitic  serum.  This  is  done  by  taking  a  series  of  dilutions, 
e.g.  from  1  in  2  to  1  in  64,  adding  to  each  of  these  hsemolytic  system 
and  complement,  and  observing  (after  incubation)  the  least  dilution 
of  antigen  which  ceases  to  fix.  This  having  been  ascertained,  this 
particular  dilution  of  antigen  is  now  tested  with  a  good  known 
positive  syphilitic  serum  to  see  that  it  does  fix  complement  under 
these  conditions.  If  the  antigen  does  not  fulfil  these  requirements 
it  must  be  rejected  and  a  fresh  one  prepared. 

(b)  Hcemolytic    system. — This  may  be  serum  haemolytic  for  ox, 
sheep,  or  human  red-blood  corpuscles,  with  the  homologous  cor- 
puscles (see  p.  183). 

The  hcemolytic  serum  or  hcemolytic  amboceptor  is  usually  prepared 
in  the  laboratory  by  injecting  a  rabbit  with  washed  red-blood 
corpuscles  (see  p.  184),  but  the  horse  is  occasionally  employed. 
The  haemolytic  serum  should  be  of  high  titre  and  may  conveniently 
be  stored  in  quill-tubing  or  in  small  ampoules,  which  after  sealing 
are  heated  in  a  water-bath  to  57°  C.  for  an  hour  on  three  or  four 
successive  days  and  kept  in  a  dark  cool  place.  This  sterilises  and 
destroys  the  complement,  leaving  only  the  haemolytic  amboceptor. 

The  blood  corpuscles. — If  blood  is  obtained  from  the  slaughter- 
house it  should  be  defibrinated  at  the  time  of  bleeding.  If  human 
blood  is  used  (Noguchi's  method),  the  blood  from  a  prick  is  allowed 
to  drip  into  citrated  saline  solution.  In  either  case,  the  corpuscles 
are  washed  twice  with  0-85  saline  solution  and  a  sufficiency  is  used 
to  make  a  5  per  cent,  suspension  by  volume  in  the  saline  solution. 
This  is  preferably  used  fresh,  but  will  keep  for  a  day  or  two  in  the 
ice-safe. 

(c)  Complement. — Fresh  guinea-pig  serum  diluted  to  1  in  10  with 
0-85  per  cent,  saline  is  usually  employed,  though  in  some  methods 
the  complement  present  in  the  serum  to  be  tested  is  made  use  of. 
In  the  method  here  described  the  amount  of  complement  present 
in  the  fresh  guinea-pig's  serum  is  ascertained,  the  serum  being  then 
diluted  to  double  the  minimum  amount  required  to  produce  complete 
haemolysis. 

Every  fresh  lot  of  the  hsemolytic  amboceptor  should  be  tested 
both  as  to  its  haemolytic  activity  and  also  as  to  the  amount  of 
complement  necessary  to  produce  complete  haemolysis  with  varying 


504 


A  MANUAL  OF  BACTERIOLOGY 


TABLE  A. 

TABLE  illustrating  the  manner  of  testing  the  activity  of  hsemolytic 
amboceptor. 


5  per 

Amboceptor 

Guinea- 

cent,  sus- 

dilution 
Amount  in 
each  test 

pig  com- 
plement 
in  50  per 

pension 
of 
sheep's 

Salt 
solution. 

Amount  of 
amboceptor 
used 

Dilution 
of  ambo- 
ceptor 

Result. 
Amount  of 
Haemolysis 

0-1  c.c.) 

cent.  sol. 

cor- 

puscles 

1:10 

0-1  c.c. 

0-5  c.c 

1-8  c.c. 

0-01  c.c. 

250 

+  +  +  + 

1:20 

0-1  c.c. 

0-5  c.c. 

1-8  c.c. 

0-005  c.c. 

500 

+  +  +  + 

1  :50 

0-1  c.c. 

0-5  c.c. 

1-8  c.c. 

0-002  c.c. 

1250 

+  + 

1  :70 

0-1  c.c. 

0-5  c.c. 

1'8  c.c. 

0-00142  c.c. 

1750 

-f  + 

1  :100 

0  1  c.c. 

0-5  c.c. 

1-8  c.c. 

0-001  c.c. 

2500 

+ 

1:200 

0-1  c.c. 

0-5  c.c. 

1-8  c.c. 

0-0005  c.c. 

5000 

+ 

Control  (with- 

out ambo- 

ceptor) 

0-1  c.c. 

0-5  c.c. 

1-9  c.c. 

— 

— 

0 

Three  to  four  times  the  minimum  complete  haemolytic  dose  of 
hsemolytic  serum  is  employed  in  the  test,  viz.  in  the  above  test  where 
a  dilution  of  the  amboceptor  of  1  in  500  is  the  minimum  quantity 
showing  complete  haemolysis,  a  dilution  of  about  1  in  150  would  be 
used. 

TABLE  B. 

TABLE  illustrating  the  manner  of  testing  the  amount  of  complement 
necessary  to  produce  complete  haemolysis  in  the  presence  of 
varying  amounts  of  amboceptor. 


0-5  c.c.  of  a  5  per 

cent,  suspension 

Results. 

of  Sheep's 

Amboceptor  diluted 

corpuscles 
sensitised  with 

Undiluted 
complement 

0-05  c.c.  of  the 

diluted 

amboceptor 

1:5 

1:10 

1  :45 

0-55  c.c. 

0-15  c.c. 

+   +   +   + 

+    4-4-4- 

+  +  +  + 

0-55  c.c. 

0-12  c.c. 

+   +   -f   + 

+    +    +    + 

+    4-4-4- 

0-55  c.c. 

0-10  c.c. 

+  +  +   + 

4-    +    +    + 

+    4-    + 

0-55  c.c. 

0-08  c.c. 

+   +   +   + 

+    +    + 

+    + 

0-55  c.c. 

0-05  c.c. 

+  + 

+ 

+ 

(The  number  of  +  signs  indicates  the  amount  of  haemolysis ; 
+  +  +  +  =  complete,  4-  4-  +  =  nearly  complete,  +  +  —  partial, 
+  =  trace.) 


THE  WASSERMANN  REACTION  505 

amounts  of  the  hsemolytic  amboceptor.  In  this  way  the  haemolytic 
amboceptor  is  standardised  and  the  manner  of  carrying  out  these 
two  tests  is  illustrated  by  the  Tables  (A  and  B)  on  p.  504. 

(d)  Fluid  to  be  tested. — Either  the  blood-serum  or  the  cerebro- 
spinal  fluid  is  used  in  the  test.  If  the  test  is  carried  out  in  small 
test  tubes,  then  5  c.c.-lO  c.c.  of  blood  must  be  withdrawn  from  a 
superficial  vein  with  a  sterile  10  c.c.  syringe.  If,  however,  the  test 
is  carried  out  in  small  quill -tubes  (which  the  writer  believes  is  quite 
as  efficient  as  with  larger  tubes),  then  only  1  c.c.  or  2  c.c.  of  blood 
or  less  are  needed  (a  Wright's  capsule-full  will  suffice),  and  this  may 
be  obtained  from  the  ear  or  by  binding  some  small  rubber  tubing 
round  the  thumb  and  puncturing  the  soft  tissues  at  the  side  near 
the  nail :  when  bleeding  ceases,  the  rubber  ligature  should  be 
removed  and  re -applied,  and  this  may  be  repeated  two  or  three 
times.  The  blood  should  be  collected  in  a  sterile  tube  and  allowed 
to  coagulate  ;  this  may  be  hastened  if  necessary  by  placing  the 
tube  of  blood  in  the  warm  incubator  for  half  an  hour  and  centri- 
fuging.  After  the  serum  is  separated,  it  is  pipetted  into  another 
tube,  which  is  then  heated  in  a  water- bath  to  56°  C.  for  half  an 
hour  immediately  before  testing  in  order  to  destroy  its  content  of 
complement.  The  latter  procedure  is  important  as  a  proportion 
of  sera  from  diseases  other  than  syphilis  may  react  positively  if 
un  heated. 

In  certain  nervous  diseases,  e.g.  general  paralysis  and  tabes,  it 
may  be  necessary  to  test  the  cere bro -spinal  fluid,  which  may  react 
positively  when  the  serum  is  negative.  The  fluid  is  obtained  by 
lumbar  puncture.  An  amount  rather  larger  than  the  serum  is 
required  ;  it  should  be  free  from  blood  and  cellular  elements  (which 
may  be  removed  by  centrifuging  if  necessary)  and  it  should  not  be 
heated. 

THE  TEST. — Tubes  about  3  in.  by  -^  in.  diameter  are  used  when 
the  "  large  quantity  "  method  is  carried  out  and  the  necessary 
quantities  of  the  reagents  are  measured  with  1  c.c.  pipettes  divided 
into  hundredths.  In  the  "  small  quantity  "  method  tubes  about 
1|  in.  by  i  in.  internal  diameter  are  used,  and  the  necessary  quan- 
tities are  measured  with  a  Wright's  pipette  furnished  with  a  rubber 
teat  and  having  a  volume  or  unit  marked  with  a  grease -pencil 
about  f  in.  from  the  point,  and  also  a  four -unit  mark  higher  up. 
Dilutions  are  made  with  0-85  per  cent,  saline  solution,  and  in  all 
cases  the  same  total  volume  should  be  maintained  in  all  the  tubes. 
The  "  small  quantity  "  method  may  be  now  described  ;  if  the 
"  large  quantity  "  method  is  adopted  the  principle  is  precisely 
similar  but  larger  volumes  of  the  reagents  are  used.  For  the 


506  A  MANUAL  OF  BACTERIOLOGY 

details  of  the  "small  quantity"  method  here  given  I  am  indebted 
to  my  friend  and  colleague,  Dr.  F.  E.  Taylor,  who  has  elaborated 
it  in  this  form. 

For  a  single  serum  six  small  quill  tubes  are  required,  five  being  con- 
trols and  the  sixth  the  test,  and  for  every  additional  serum  two  more 
tubes  are  required.  The  six  tubes  (and  the  additional  ones  also 
when  more  than  one  serum  is  being  tested)  are  arranged  in  two  rows 
in  a  metal  rack  which  is  immersed  in  a  water-bath  and  maintained 
throughout  the  test  at  a  temperature  of  38°-40°  C.  To  each  tube 
in  the  back  row  run  in  4  volumes  of  saline  solution  with  the  marked 
Wright's  pipette,  into  each  tube  of  the  front  row  four  volumes  of 
the  antigen  suitably  diluted  as  ascertained  by  the  standardisation 
of  the  antigen.  Then  commencing  from  the  left  hand,  add  to  each 
of  the  first  two  tubes  (back  and  front)  one  volume  of  diluted  known 
normal  inactivated  serum  (these  form  the  negative  control  mixtures). 
To  the  next  two  tubes  add  one  volume  of  diluted  known  syphilitic  in- 
activated serum  (these  form  the  positive  control  mixtures).  Then 
to  each  of  the  next  two  tubes  add  one  volume  of  the  inactivated 
serum  to  be  tested,  diluted  1  in  2,  and  repeat  this  with  as  many 
series  of  tubes  as  there  may  be  sera  to  be  tested.  Next  to  every 
tube  add  one  volume  of  suitably  diluted  complement,  and  leave  in 
the  warm  bath  for  five  minutes.  After  this  add  five  volumes  of  the 
prepared  haemolytic  system  and  leave  in  the  bath  for  fifteen  minutes. 
The  haemolytic  system  is  prepared  by  mixing  in  bulk  four  volumes 
of  suitably  diluted  inactivated  haemolytic  serum  and  one  volume  of 
20  per  cent,  suspension  of  washed  sheep's  corpuscles.  The  pipette 
used  for  the  additions  of  the  reagents  should  be  rinsed  with  saline 
solution  between  each  constituent  of  the  test. 

At  the  end  of  fifteen  minutes  the  tubes  are  centrifuged  and  the 
pressure  or  absence  of  haemolysis  noted.  If  haemolysis  has  occurred, 
the  fluid  in  the  tubes  form  a  clear  red  solution  without  deposit  of 
corpuscles,  whereas  if  fixation  is  complete  the  corpuscles  are 
deposited  at  the  bottom  of  the  tube  while  the  fluid  above  is  colourless 
and  transparent. 

All  the  tubes  in  the  back  row  should  show  haemolysis  as  they 
contain  no  antigen,  tube  1  in  the  front  row  should  also  show  haemo- 
lysis as  it  contains  antigen  and  a  negative  serum,  tube  2  in  the  front 
row  should  show  no  haemolysis  as  it  contains  both  antigen  and 
positive  serum.  In  the  remaining  front  row  tubes,  haemolysis  or 
fixation  will  occur  according  as  the  sera  are  negative  or  positive 
respectively. 

If  it  be  desired  to  obtain  some  idea  of  the  amount  of  syphilitic 
amboceptor  present  in  a  positive  serum,  a  quantitative  estimation 


INFUSORIA  507 

may  be  carried  out  by  putting  up  a  series  of  tubes  containing  either 
diminishing  quantities  of  the  serum  or  diminishing  quantities  of 
antigen,  the  other  constituents  remaining  the  same  in  either  case. 

As  human  serum  generally  contains  amboceptor  hsemolytic  for 
sheeps'  corpuscles,  Flemming  *  devised  a  method  in  which  the 
test  serum  itself  with  sheep's  corpuscles  constitutes  the  haemolytic 
system,  and  the  test  is  also  carried  out  with  Wright's  pipettes. 
Emery  2  in  his  method  makes  use  of  human  corpuscles  and  of  the 
complement  present  in  the  test  serum  so  that  addition  of  com- 
plement is  unnecessary  (in  this  case,  of  course,  the  test -serum  is 
not  inactivated). 

The  examination  of  a  very  large  number  of  cases  of  syphilis  by 
different  observers  indicates  that  the  test  is  of  very  considerable 
value  and  diagnostic  significance.  In  conditions  such  as  tabes 
dorsalis  and  general  paralysis  of  the  insane,  which  on  other  grounds 
are  generally  regarded  as  due  to  syphilis,  52  per  cent,  give  the 
reaction.  A  positive  reaction  may  be  said  to  show  a  positive,  and 
probably  active,  syphilitic  infection,  but  a  negative  reaction  does 
not  necessarily  exclude  syphilis.  Energetic  mercurial  treatment 
may  render  the  reaction  negative. 

(4)  Forges'  reaction. — If  syphilitic  serum  be  added  to  a  solution 
of  lecithin  or  other  lipoid  substances,  in  many  cases  it  gives  a  white 
precipitate.  Normal  or  non-syphilitic  serum  gives  no  precipitate. 
This  has  been  tried  extensively  as  a  substitute  for  the  Wassermann 
reaction,  but  it  is  not  so  delicate. 


Class  III. — Infusoria  (Ciliata) 

The  Infusoria  are  protozoa  the  locomotive  organs  of  which 
consist  of  cilia,  and  in  which  the  nuclear  apparatus  is  differentiated 
into  a  vegetative  macronucleus  and  a  generative  micronucleus. 
The  cytoplasm  is  enclosed  within  a  cuticle,  an  oral  aperture  is 
present  in  the  form  of  a  slit  or  pore,  and  waste  matter  is  extruded 
by  a  pore,  constant  in  position,  but,  as  a  rule,  visible  only  when  in 
use.  A  contractile  vacuole  is  generally  present.  Reproduction 
usually  takes  place  by  fission,  which  is  preceded  by  division  of  the 
two  nuclei,  the  micronucleus  by  mitosis,  the  macronucleus  by  direct 
division. 

The  Infusoria  are  not  of  much  pathological  importance,  but  are 
common  in  ponds  and  ditches,  e.g.  Paramecium  and  Vorticella. 

1  Lancet,  1909,  vol.  i,  p.  1512. 

2  Ibid.  1910,  vol.  ii,  September  3. 


508 


A  MANUAL  OF  BACTERIOLOGY 


Balantidium  (Paramecium)  coli 

This  is  an  intestinal  parasite  of  swine,  occasionally  met  with  in 

man  in  conditions  associated  with  chronic  diarrhoea  and  dysentery. 
It  is  somewhat  ovoid  in  shape,  the  ends  being  bluntly  pointed,  is 
covered  with  cilia,  measures  65  to  85  /x  in 
length,  and  has  a  superficial  resemblance 
to  the  ordinary  Paramecium. 

According  to  Saville  Kent,  the  Balan- 
tidium coli  is  to  be  distinguished  from  the 
ordinary  forms  of  water  paramecia  by  the 
following  characters  :  The  Bal.  coli  is  some- 
what spindle-shaped  or  ovoid,  and  bluntly 
pointed  at  each  end,  one  and  a  half  to 
twice  as  long  as  broad,  measuring  ^i^  in. 
to  T  J-^  in.  in  length  ;  the  paramecium  is 
more  cylindrical,  four  times  as  long  as 
broad,  measuring  yl^  in.  to  ¥TF  in.  in 
length.  The  oral  aperture  in  Bal.  coli  is 
near  one  extremity  (Fig.  57)  ;  in  para- 
mecium it  is  situated  at  about  the  middle 
of  the  ventral  surface.  In  Bal.  coli  the 
cilia  round  the  oral  aperture  are  as  long 
again  as  those  over  the  body  generally ;  in 

paramecium  the  whole  of  the  cilia  are  of  the  same  length. 

The  Bal.  coli  seems  undoubtedly  sometimes  to  be  a  cause  of 

dysentery.1     Bal.  coli  is  a  common    parasite   of   pigs   and   may 

contract  infection  from  these  animals. 


FIG.  57. — Balantidium 
coli. 


Examination  of  Flagellated  and 
Ciliated  Forms 

(1)  These  may  be  examined  fresh  in  the  fluid  in  which  they  are 
present,  by  mounting  on  a  slide,  and  covering  with  a  cover-glass 
one  edge  of  which  rests  on  a  bristle  to  avoid  pressure. 

(2)  Permanent  mounts  may  be  made  by  the  Heidenhain  method 
(p.  485). 

(3)  Films  may  be  made  in  the  ordinary  way,  and  stained  with 
weak   carbol-fuchsin    or   Leishman's   stain.     (The    organisms    are 
apt  to  be  distorted. ) 

(4)  The  following  method,  devised  by  Rousselet  (Journ.  Quekett 

1  Strong  and  Musgrave,  Johns  Hopkins  Hosp.  Ball.,  vol.  xii,  1901, 
p.  31  ;   Bureau  of  Gov.  Laboratories,  Manila,  Bull.  26,  1904. 


COCCIDIA  509 

Microscop.  Club,  2nd  series,  vol.  vi,  no.  36,  p.  5,  March,  1895)  for 
preserving  Rotatoria,  may  be  tried.  In  those  forms  which  are 
non-contractile,  kill  by  adding  a  drop  of  J  per  cent,  osmic  acid, 
wash  immediately  in  water,  and  preserve  in  2£  per  cent,  formalin. 
Contractile  forms  may  be  first  narcotised  by  adding  a  drop  or  two 
of  2  per  cent,  cocaine  solution,  then  killed  with  the  osmic  and 
preserved  as  before. 


Class  IV. — Sporozoa 

The  sporozoa  are  exclusively  endoparasitic  protozoa,  the  adult 
lacking  organs  for  locomotion  and  for  the  capture  of  food,  and 
multiply  by  some  method  of  sporulation,  often  very  complex. 
Binary  fission  is  almost  unknown  in  this  group.  A  parasite  during 
the  nutritive  or  "  trophic  "  phase,  when  it  is  absorbing  nutri- 
ment and  growing  at  the  expense  of  its  host,  is  termed  a  trophozoite  ; 
when  it  is  mature  and  ready  for  sporulation  it  is  termed  a  sporozoite 
or  schizont.  The  spores  are  of  various  kinds,  and  may  develop 
outside  the  body  or  in  a  second  host. 


Order. — Coccidiidea 

The  Coccidiidea,  with  a  single  exception,  are  intra-cellular  during 
the  trophic  stage,  and  present  a  dimorphism  or  alternation  of 
generations  ;  the  one  is  endogenous  and  asporular,  determining 
the  reproduction  of  the  parasite  within  the  host,  the  other  exogenous 
and  sporular  and  permitting  of  infection. 

Coccidial  Disease  of  Rabbits 

This  is  a  disease  caused  by  a  sporozoon,  the  Coccidium  (Eimeria) 
oviforme  or  cuniculi,  and  often  met  with  in  warrens  and  hutches  ; 
in  some  of  the  former  as  many  as  90  per  cent,  of  the  animals  may  be 
affected.  The  young  animals  suffer  most,  and  become  infected  when 
they  cease  to  suckle  and  commence  to  eat  green  food,  the  adult  ani- 
mal as  a  rule  resisting  the  disease.  The  affected  animals  waste,  suffer 
from  enteritis,  and  a  large  proportion  die  in  from  one  to  three  weeks, 
the  condition  being  known  as  "  wet-snout  "  among  the  keepers. 
The  parasites  occur  in  the  intestine,  bile-ducts,  and  liver  in  large 
numbers.  Each  parasite  is  ovoid  in  shape,  measuring  36  p.  in  length 
and  22  p,  in  breadth,  is  enclosed  in  a  firm  translucent  cyst,  which 
encircles  a  very  granular  protoplasm.  Sometimes  this  protoplasm 


510 


A  MANUAL  OF  BACTERIOLOGY 


becomes  condensed  so  as  to  form  a  spherical  mass  lying  free  within 
the  cyst  (Fig.  58,  A).  In  the  intestine  and  bile-ducts  the  parasites 
are  attached  to  the  epithelial  cells,  and  in  the  liver,  if  the  animal 
lives  beyond  the  acute  stage,  set  up  some  remarkable  changes.  The 
affected  liver  is  studded  with  greyish-white  nodules  varying  in 


FIG.  58. — Diagram  of  Development  of  Coccidia.1 

size  from  a  pin's  head  to  a  pea.  On  making  sections  and  examining 
them  microscopically,  it  is  found  that  these  nodules  consist  of 
dilated  bile-ducts  filled  with  a  much  hypertrophied  and  convoluted 
mucous  membrane,  which  forms  branched  projections  covered  with 
cubical  epithelium,  among  which  the  parasites  occur  in  great  numbers 
(Plate  XXIII.  b).  A  curious  fact  is  that  subcutaneous  or  intra- 
venous inoculation,  or  inoculation  into  the  liver  of  a  healthy  rabbit 
with  the  coccidia  from  another  rabbit,  fails  to  induce  the  disease. 

1  This  diagram  is  reproduced  by  permission  from  Daniel's  Tropical 
Medicine  and  Hygiene,  2nd  ed.  1913  (John  Bale,  Sons,  and  Danielsson). 


COCCIDIA  511 

The  coccidium  has  a  complicated  developmental  history,  and 
infection  only  seems  possible  in  one  of  the  stages.  In  order  to  study 
the  life -cycle  the  parasite  must  be  placed  under  suitable  conditions, 
and  an  infusion  of  rabbits'  faeces,  kept  at  the  ordinary  temperature, 
is  perhaps  as  good  a  cultivating  medium  as  any,  the  changes  being 
watched  by  means  of  interlamellar  films.  Reproduction  may  be 
either  asexual  or  sexual,  and  may  be  endogenous,  within  the  host, 
or  exogenous,  outside  the  host.  In  the  asexual  cycle,  division  of 
the  protoplasm  and  nucleus  of  the  coccidium  takes  place  and  the 
cyst  comes  to  contain  large  numbers  of  spores  (Fig.  58,  A).  The 
cyst-wall  then  ruptures,  the  spores  are  liberated,  pass  into  other 
intestinal  or  hepatic  cells  and  reproduce  the  coccidium  once  more 
(Fig.  58,  A).  In  the  sexual  cycle,  the  protoplasm  of  some  coccidia 
remains  undivided  with  a  single  nucleus  and  the  cyst  has  a  weak 
spot,  known  as  the  micropyle  ;  these  are  the  female  cells  or  macro- 
gametes  (Fig.  58,  B).  In  other  coccidia,  the  protoplasm  having 
attained  maximum  growth,  divides  into  a  mass  of  actively  motile 
thread-like  bodies,  the  male  elements  or  microgametes.  The  cyst- 
wall  then  ruptures  and  the  microgametes,  penetrating  the  micropyle 
of  the  macrogametes,  fertilize  them.  In  the  fertilised  macrogamete, 
which  is  a  zygote  known  as  an  "  oocyst  "  and  is  non-motile,  the 
micropyle  closes  and  the  cyst  is  discharged  with  the  faeces  of  the 
animal.  On  damp  ground,  the  nucleus  and  protoplasm  divide 
into  four  spherules.  Each  spherule  becomes  elongated,  and  again 
divides  into  two  somewhat  crescent-shaped  bodies,  around  each 
pair  of  which  a  new,  somewhat  spindle-shaped  capsule  forms  (Fig. 
58,  D).  In  this  condition  the  parasite  is  very  resistant,  and  may 
remain  alive  for  six  months,  undergoing  no  further  change  unless 
introduced  into  another  animal.  If  a  young  rabbit  swallows  with 
its  food  these  crescentic  spores,  the  enclosing  capsule  is  dissolved, 
and  each  crescent  becomes  a  rounded  amoeboid  mass,  and  this 
again  divides  up  into  many  crescentic  spores.  These  spores  are 
apparently  motile,  and  enter  the  epithelial  cells  of  the  intestine, 
gall-bladder,  and  bile-ducts,  where  a  process  of  growth  and 
differentiation  occurs,  and  the  fully  developed  parasite  is  ultimately 
reproduced. 

Coccidial  disease,  or,  as  it  is  sometimes  termed,  psorospermosis, 
is  occasionally  met  with  in  animals,  as  the  sheep,  and  a  wasting 
disease  of  young  pheasants  due  to  coccidia  has  been  described  by 
McFadyean.1  Coccidiosis  also  occurs  in  grouse  and  poultry,  due 
to  Eimeria  avium ;  in  the  latter  causing  "  scour,"  which  may  be 
attended  with  considerable  loss. 

1  Journ.  Comp.  Path,  and  Therapeut.,  1895. 


512  A  MANUAL  OF  BACTERIOLOGY 

In  man,  coccidial  disease  has  been  described  (but  rarely)  in  the 
liver,  gall-bladder,  ureter,  etc.1 

Rixford  and  Gilchrist  2  described  two  cases  of  protozoan  infection 
of  the  skin  and  organs,  accompanied  by  great  destruction  of  tissue 
and  ending  in  death.  The  organisms  were  spherical,  1  to  21  p 
diameter,  surrounded  by  a  thick  capsule,  enclosing  granular  bioplasm 
(C.  immitis). 

The  Ruffer-Plimmer  bodies  of  cancer  were  at  one  time  believed 
to  be  coccidia  (p.  554). 

The  term  "  psorospermosis  "  has  been  applied  to  human  infection 
with  coccidium,  Sarcosporidia  (p.  532),  etc. 


Examination 

(1)  The  coccidial  forms  are  readily  examined  in  the  fresh  state- 
The  only  bodies  they  are  likely  to  be  mistaken  for  are  certain  ova. 

(2)  Paraffin  sections  of  rabbit's  liver  containing  coccidia  may 
be  stained  much  in  the  same  way  as  tuberculous  tissues — viz.  warm 
carbol-fuchsin  ten  minutes,   decolorise  cautiously  in   5   per  cent, 
acid,  and  counter-stain  in  methylene-blue.     Sections  may  also  be 
stained  in  the  Ehrlich-Biondi  stain  for  one  to  two  hours. 


Order. — Haemosporidia 

The  general  characters  of  this  group  are  : 

(1)  Life  at  the  expense  of  the  red  blood-corpuscles,   at  least 
during  a  portion  of  the  life-cycle. 

(2)  Endogenous   multiplication   by   spores,    by   which   the   life- 
cycle  is  repeated  within  the  host. 

(3)  Development  of  a  form  which  becomes  free  in  the  plasma, 
and  which  is  the  commencement  of  a  sexual  cycle  to  be  completed 
in  a  second  host. 

(4)  Inoculability,  but  only  from  one  animal  to  another  of  the 
same  species. 

The  group  includes  the  malaria  parasite  and  similar  parasites  in 
mammals  and  birds,  the  haemogregarines,  Drepanidium  of  the 
frog,  and  perhaps  the  Piroplasmata. 

1  Journ.  Comp.  Path,  and  Bact.,  1898,  June,  p.  171. 

2  Johns  Hopkins  Hosp.  Reps.,  vol.  i,  1896,  p.  209. 


MALARIA  513 

Malaria 

Malaria  is  caused  by  parasitic  protozoa,  placed  in  the 
genus  Plasmodium  (Hcemamceba),  the  credit  of  the  discovery 
of  which  must  be  given  to  Laveran,  who  described  the 
parasite  as  occurring  in  four  phases,  viz.  (1)  spherical 
bodies,  (2)  flagellated  bodies,  (3)  crescentic  bodies,  and 
(4)  segmented  or  rosette  bodies. 

The  parasites  cannot  be  cultivated  beyond  one 
generation,  but  inoculation  of  healthy  individuals  with 
the  blood  of  malarial  patients  reproduces  the  disease, 
and  the  same  structures  or  parasites  are  found  in  the  blood 
of  these  infected  persons.  Inoculation  experiments  on  all 
animals  except  man  have  proved  negative,  and  in  the 
latter  the  inoculation  must  be  intravenous. 

In  the  various  forms  of  malarial  fever  the  parasites  have 
the  same  general  characters,  though  there  are  distinct 
differences  between  them,  by  which  they  can  be  recog- 
nised and  the  type  of  fever  differentiated.  In  each  there 
is  an  endo-corporeal  cycle  within  the  host,  through  which 
the  recurrent  attacks  are  developed  ;  there  is  also  an 
extra-corporeal  cycle  of  development  outside  the  body  of 
the  host,  whereby  the  infection  of  fresh  individuals  becomes 
possible.  Each  of  these  cycles  needs  separate  description. 

If  the  blood  of  a  malarial  patient  is  examined  an  hour 
or  two  before,  or  at  the  very  commencement  of,  the  febrile 
paroxysm,  the  parasite  will  be  recognised  as  a  pale,  ill- 
defined  mass  of  protoplasm  within  the  red  corpuscles,  of 
which  a  variable  proportion  are  infected,  the  size  of  the 
parasite  varying  in  the  different  types  of  fever.  When 
some  hours  old  a  variable  number  of  blackish  pigment- 
granules  of  melanin  make  their  appearance.  These  subse- 
quently coalesce  into  smaller  groups,  and  the  latter  again 
into  one  or  two  larger,  more  or  less  centrally  disposed, 
masses.  The  parasites  exhibit  more  or  less  amoeboid 

33 


514  A  MANUAL  OF  BACTERIOLOGY 

movement,  and  the  melanin  granules  are  frequently  in  a 
state  of  tremor.  Later  on  most  of  the  parasites  (now 
schizonts)  become  divided  into  a  variable  number  of 
segments,  which  separate  and  become  spherical,  the  blood- 
corpuscle  breaks  down,  the  spherical  bodies  or  spores  are 
set  free,  and  a  certain  number  of  them,  again  becoming 
attached  to  red  corpuscles,  develop  into  the  first  stage  of 
the  parasite.  The  melanin  granules  and  some  of  the 
spores  are  ingested  by  phagocytes,  and  after  some  time  the 
melanin  is  deposited  in  the  spleen  and  liver. 

The  parasite,  termed  a  plasmodium,  or  better,  an  amce- 
bula,  contains  a  vesicular  nucleus  and  a  nucleolus,  and 
the  melanin  granules  are  present  in  the  surrounding  proto- 
plasm. When  segmentation  occurs,  each  segment  contains 
a  portion  of  both  the  nucleolus  and  the  protoplasm.  The 
maturation  of  each  "  brood  "  of  parasites  is  coincident 
with  a  fresh  febrile  paroxysm.  In  the  subtertian  (per- 
nicious) forms  of  malarial  fever  there  exist  in  the  blood 
for  some  time  after  the  subsidence  of  the  acute  paroxysms 
well-marked  non-motile,  crescentic  or  sausage-shaped 
bodies,  with  rounded  ends,  the  so-called  "  crescentic 
bodies  "  or  "  crescents  "  ;  their  longer  diameter  is  greater 
(^)  than  that  of  a  red  corpuscle,  their  protoplasm  is  finely 
granular,  and  contains  at  about  the  centre  several  well- 
marked  pigment-granules.  In  the  crescentic  forms  the 
extremities  of  the  crescent  often  appear  to  be  joined  by  a 
delicate  membrane  (Fig.  64,  /  and  j)  ;  this  is  the  remains 
of  the  blood- corpuscle  in  which  the  parasite  has  developed. 

When  a  "  wet "  specimen  of  malarial  blood  from  a 
case  of  pernicious  or  sub-tertian  malaria  is  kept  under 
observation  (p.  523),  it  not  infrequently  happens  that 
after  a  time  the  so-called  flagellated  "  bodies  "  make  their 
appearance.  These  consist  of  a  central  protoplasmic  mass 
attached  to  which  are  from  one  to  six  delicate  flagella 
measuring  20-30  jm  in  length  (Fig.  59,  c).  The  flagella 


THE  MALARIA  PARASITE 


515 


are  actively  motile  and  disturb  the  corpuscles,  but  the 
body  itself  does  not  move  much.  Frequently  one  or 
more  of  the  flagella  break  away  and  swim  free,  remaining 
active  for  several  hours.  The  flagellated  bodies  are  never 
seen  in  the  freshly  drawn  blood,  and  Ross  has  found  that 
flagellation  does  not  occur  if  the  finger  be  pricked  through 
a  spot  of  vaseline,  the  blood  remaining  covered  with  the 


FIG.  59. — Development  of  the  malaria  parasite  in  the  mosquito. 
a,  b,  and  c,  the  male  gametocyte  ;  d,  e,  and  /,  the  female 
gametocyte ;  /,  fertilisation  of  the  female  gametocyte  by  a 
microgamete.  (After  Ross  and  Fielding- Ould.) 

film  of  grease.  Careful  observation  has  shown  that  the 
flagellated  bodies  develop  from  "  crescents  "  in  subtertian 
malaria,  and  from  special  rounded  parasites,  difficult  to 
distinguish  from  the  schizonts,  in  the  benign  tertian  and 
quartan  fevers. 

Various  theories  were  held  in  the  past  as  to  the  nature 
of  these  flagellated  bodies.  Through  the  brilliant  researches 
of  Ross,  which  have  been  confirmed  and  extended  by 
observers  in  all  parts  of  the  world,  it  is  now  known  that 
these  cells  are  sexual  elements.  The  flagellated  body 
represents  the  male  cell  or  "  male  gametocyte,"  the  flagella 
("  gametes ")  being  analogous  to  the  spermatozoa  of 


516  A  MANUAL  OF  BACTERIOLOGY 

higher  animals.  The  female  cells  or  female  gametocytes 
or  gametes  are  non-flagellated,  and  are  fertilised  by  the 
entrance  of  one  of  the  flagella  of  a  male  gametocyte.  This 
fertilisation  takes  place  in  the  stomach  (middle  intestine)  of 
certain  species  of  mosquito,  and  after  fertilisation  a  series  of 
changes  ensues  resulting  in  the  formation  of  spore-like 
bodies,  which  are  injected  when  the  insect  bites  its  victim, 
and  thus  the  infection  of  fresh  individuals  with  the  malaria 
parasite  takes  place.  The  first  demonstration  of  the  nature 
of  "  flagellated  bodies  "  was  given  by  Opie  and  MacCallum 
on  the  Halteridium,  a  parasite  of  pigeons  (p.  528),  and  this 
forms  a  good  example  of  the  value  of  abstract  research 
to  practical  medicine  (see  p.  528).  Ross  also  followed  the 
development  of  the  malaria-like  Proteosoma  of  sparrows, 
etc.,  in  the  mosquito,  Culex  fatigans.  The  development 
of  the  malaria  parasite  of  man  in  the  mosquito  is  as  follows, 
according  to  Ross  and  Fielding- Ould.1  It  is  not  known 
what  determines  whether  an  amoebula  will  become  a 
sporocyte  or  a  gametocyte.  When  the  sexual  cells  or 
"  gametocytes  "  are  ingested  with  the  blood  by  the  mos- 
quito, they  pass  into  the  middle  intestine.  Within  a  few 
minutes  the  corpuscles  enclosing  them  break  down,  the 
parasites  are  set  free,  and  quickly  become  spherical  or 
ovoid  (Fig.  59,  c,  e,  and/).  One  or  two  spherical  granules 
are  often  attached  to  the  naked  parasites,  and  may  repre- 
sent polar  bodies  (Fig.  59,  c  and  /).  Very  soon  the  male 
cells  become  flagellated  (Fig.  59,  c),  and  before  long  the 
flagella  or  "  microgametes  "  break  away  from  the  parent 
cell,  and  by  their  own  motility  make  their  way  through 
the  liquor  sanguinis.  Should  one  come  in  contact  with 
a  female  cell  or  "  macrogamete,"  it  fuses  with  the  latter, 
uniting  with  the  nucleus  (Fig.  59,  /),  fertilisation  is  com- 
pleted, and  a  "  zygote  "  is  formed.  As  the  zygote  at  this 
stage  is  motile  it  is  known  as  a  "  travelling  vermicule  "  or 

*  Thompson  Yates  Laboratories  Report,  vol.  iii,  pt.  ii,  p.  183. 


THE  MALARIA  PARASITE 


517 


"  ookinet  "  ;  it  passes  into  the  outer  wall  of  the  mosquito's 
stomach,  where  it  becomes  encysted  (Fig.  60,  a,  6).  At 
this  period  the  zygote  is  about  7-8  ^  in  diameter.  If 
development  proceeds,  it  acquires  a  distinct  capsule  and 
begins  to  grow  rapidly,  and  when  mature  at  the  end  of  a 


FIG.  60. — Development  of  the  malaria  parasite  in  the  mosquito. 
(After  Ross  and  Fielding-Ould.) 

week  or  more,  according  to  the  temperature,  is  60  /x  in 
diameter,  and  projects  into  the  body-cavity  of  the  insect 
(Fig.  60,  b).  Its  substance  next  divides  into  eight  to 
twelve  portions,  or  "  zygotomeres,"  then  each  zygotomere 
becomes  a  spherical  body,  or  "  blastophore  "  (Fig.  60,  c), 
and  each  blastophore  develops  upon  its  surface  a  number 
of  spindle-shaped,  radially  disposed  bodies,  or  "  zygoto- 
blasts  "  (Fig.  60,  d).  When  the  zygote  reaches  maturity 


518 


A  MANUAL  OF  BACTERIOLOGY 


the  blastophores  disappear,  leaving  its  capsule  packed  with 
large  numbers  ("  thousands  ")  of  free  zygotoblasts.  The 
capsule  then  ruptures,  and  the  zygotoblasts  are  poured 
into  the  body-cavity  of  the  mosquito.  The  "  blasts " 
measure  12-16  JUL  in  length,  taper  at  each  extremity,  and 


The 
Mosquito  Phase 

Exogenous 
,or  Sexual  Cycle 


FIG.  61. — Diagram  of  the  asexual  and  sexual  cycles  of  the 
malaria  parasite. 


possess  a  central  nucleus  (Fig.  60,  e),  and  they  make  their 
way  to  all  parts  of  the  body  of  the  host,  and  accumulate 
in  the  salivary  or  poison  glands,  whence  they  are  dis- 
charged by  the  middle  stylet  (hypopharynx)  of  the  pro- 
boscis, when  the  insect  "  bites,"  into  the  circulation  of  a 
fresh  vertebrate  host.  Here,  presumably,  the  blasts  be- 
come attached  to  erythrocytes  and  develop  into  amcebulse. 


THE  MALARIA  PARASITE  519 

The  diagram1  (Fig.  61)  represents  in  graphic  form  the  asexual 
and  sexual  cycles  of  reproduction  of  the  malaria  parasite. 
So  far  as  is  known,  malarial  infection  is  conveyed  only 
through  the  bite  of  infected  mosquitoes  of  the  sub-family 
Anophelince.  It  has  been  repeatedly  proved  that  infected 
mosquitoes  convey  infection,  and  that  if  mosquitoes  be 
excluded  human  beings  may  live  in  the  most  malarious 
districts  without  contracting  the  disease. 

Mosquitoes  (Culicidce)  are  distinguished  from  other  mosquito- 
like  insects  by  the  fringe  of  scales  on  the  wings.  The  common 
mosquitoes  belong  to  the  sub-family  Culicince.  The  Anophelince, 
are  usually  less  abundant  (but  there  is  great  variation  in  different 
districts),  and  bite  mainly  at  night ;  the  females  alone  are  blood- 
suckers. Some  species  breed  in  natural  collections  of  stagnant, 
others  in  slowly  running  fresh,  water  well  supplied  with  lowly  forms 
of  vegetable  life.  If  the  head  of  a  mosquito  be  examined  with  a 
hand-lens,  three  sets  of  appendages  will  be  noticed.  In  the  middle 
is  the  stout  proboscis  containing  the  stinging  and  suctorial  appa- 
ratus ;  situated  at  the  base  of  this  are  two  palpi,  one  on  either  side, 
and  outside  these  again  are  two  antennae,  which  are  more  or  less 
hairy.  In  Anophelince,  both  male  and  female,  the  palpi  are  as  long 
as  the  proboscis  ;  in  the  female  Culex  (also  in  Stegomyia  and  many 
other  genera)  they  are  short  and  stumpy.  In  Anophelince  the  scales 
on  the  veins  of  the  wings  are  usually  arranged  in  alternating  light 
and  dark  patches,  giving  a  speckled  or  dappled  appearance,  different 
as  a  rule  from  anything  seen  in  Culex.  (Some  Culices  have  a  similar 
arrangement,  and  it  is  wanting  in  A.  maculipennis  and  A.  bifurcatus.) 
The  front  or  costal  margin  of  the  wing  in  Anophelince  is  almost 
always  marked  with  dark  blotches.  Anopheles,  as  a  whole,  is  a 
more  slender  insect  than  Culex,  and  when  at  rest  its  body  is  all  in 
one  line,  whereas  Culex  is  angular  or  hump-backed.  The  important 
species  known  to  carry  malaria  are  Anopheles  maculipennis  in  Europe, 
N.  Africa,  and  N.  America,  A.  bifurcatus  in  Europe,  Myzomyia 
funesta  and  Pyretophorus  costalis  in  Central  and  W.  Africa,  and 
Cellia  argyrotarsis  in  tropical  America.  Other  species,  e.g.  Myzo- 
rhynchus  sinensis,  Cellia  Kochii,  and  others,  are  less  important 
carriers. 

(On  Mosquitoes,  see  Theobald,  Brit.  Museum  Monograph,  and 
Allbutt's  System  of  Med.,  ed.  2,  vol.  ii,  pt.  2  ;  Giles,  Handbook  of 

1  This  figure  is  reproduced  by  permission  from  Daniels'  Laboratory 
Studies  in  Tropical  Medicine  (Bale,  Sons,  &  Danielsson,  1908). 


520 


A  MANUAL  OF  BACTERIOLOGY 


the  Gnats  and  Mosquitoes ;  Daniels,  Laboratory  Studies  in  Tropical 
Medicine,  ed.  3,  1908.) 

There  are  probably  at  least  three  species  of  malaria 
parasite  l  occurring  in  the  various  types  of  malarial  fever 
in  man,  though  some  authorities  (e.g.  Laveran)  regards 
the  forms  as  varieties  of  a  single  species,  and  the  following 
are  the  differential  characters  between  them  : 

(1)  Benign  quartan  fever  (Fig.  62). — The  quartan  parasite 


FIG.  62. — The  quartan  parasite  :  a,  6,  c,  d,  amoebulse ;  e, 
sporocyte ;  /,  free  spores ;  g,  female  gametocyte  with  so- 
called  polar  body  ;  h,  male  gametocyte.  (After  Rees.) 

(Plasmodium  malarice)  completes  its  asexual  life- cycle 
in  seventy- two  hours  ;  there  are  two  complete  days  without 
an  attack,  and  reckoning  the  day  of  the  previous  attack, 
an  attack  occurs  every  fourth  day,  hence  the  name  "  quar- 
tan." It  commences  as  a  small  amcebula,  which  is  feebly 
motile.  It  enlarges,  becomes  pigmented,  and  motility 
ceases,  the  pigment-granules  being  numerous  and  coarse. 
The  parasite  finally  occupies  nearly  the  whole  of  the 
corpuscle,  which,  however,  is  but  little  altered  (a-d). 

Towards  the  end  of  the  apyrexial  period  the  pigment 
collects  in  the  centre,  and  segmentation  takes  place  with 
the  formation  of  a  symmetrical  rosette  (e),  and  afterwards 
of  six  to  twelve  spores  (/).  The  quartan  parasite  does  not 

1  Hewlett,  Trans.  XlVth  Internal.  Congress  of  Hygiene,  vol.  ii.  1908, 
p.  141, 


PLATE  XXIV 


a.  Malaria. 


Parasite  of  benign  tertian  fever. 
X  1500. 


Smear  of  blood. 


b.  Malaria. 


Gametocyte  of  benign  tertian  parasite, 
of  blood.      X  1500. 


Smear 


THE  MALARIA  PARASITE 


521 


form  crescents,  and  the  flagellated  bodies  (h),  which  are 
rarely  seen,  are  developed  from  large  pigmented  parasites. 
(2)  Benign,  or  spring,  tertian  fever  (Fig.  63 ;  Plate 
XXIV.  a). — The  benign  tertian  parasite  (Plasmodium  vivax) 
completes  its  asexual  life- cycle  in  forty- eight  hours,  an 
attack  occurring  every  other  day,  or,  reckoning  the  day 
of  the  previous  attack,  every  third  day.  In  the  early 
stage  it  resembles  the  quartan,  but  shows  much  more 


FIG.  63. — The  benign  tertian  parasite  :  a,  b,  c,  d,  amcebulae  ; 
e,  sporocyte  ;  /,  free  spores  ;  g,  female  gametocyte  with  so- 
called  polar  bodies  ;  h,  male  gametocyte.  (After  Rees.) 

active  amoeboid  movement.  The  pigment-granules  are 
also  finer  than  in  the  quartan,  and  incessantly  change 
their  position.  The  parasite  finally  invades  the  whole 
corpuscle,  which  becomes  enlarged  and  pale.  Enlarge- 
ment of  the  corpuscles  is  a  marked  feature  in  the  benign 
tertian  infection  (d). 

Segmentation  takes  place,  but  is  unsymmetrical  (e), 
resulting  in  the  formation  of  a  grape-like  cluster  of  twelve 
to  twenty  spores  (/).  As  in  the  quartan,  no  crescentic 
bodies  are  developed,  and  the  gametocytes  (g,  h)  are 
similar  to,  but  larger  than,  the  quartan  (Plate  XXIV.  b). 

(3)  The  cestivo-autumnal,  malignant,  pernicious,  or  sub- 
tertian,  fevers  (Fig.  64). — This  parasite  (Laverania  malarice) 
(or  parasites,  for  it  has  been  divided  into  three  species 


522 


A  MANUAL  OF  BACTERIOLOGY 


by  the  Italian  observers,  viz.  the  pigmented  and  the 
unpigmented  quotidian  and  the  malignant  tertian,  but 
this  is  not  generally  accepted)  is  much  smaller  than  the 
quartan  or  benign  tertian,  and  when  it  reaches  the  stage 
of  multiplication  it  disappears  from  the  peripheral  blood 
and  collects  in  the  internal  organs,  spleen,  liver,  cerebral 
capillaries,  and  bone-marrow.  It  is  actively  amoeboid, 
seems  to  change  its  position  within  the  corpuscle,  and 
the  pigment-granules  are  very  fine  in  the  young  parasites, 


g  h          j 

FIG.  64. — The  sub-tertian  parasite  :  a,  b,  c,  amoebulse  ;  d,  sporo- 
cyte  ;  e,  free  spores ;  /,  g,  h,  female  gametocyte ;  j,  k,  I, 
male  gametocyte.  (After  Rees.) 

but  early  aggregate  into  large  clumps.  The  fission  forms 
(d,  e)  are  only  met  with  in  the  internal  organs.  Multiple 
infection  of  the  corpuscles  may  also  occur.  The  corpuscles 
often  suffer  severely  from  the  infection,  some  being  shrivelled 
and  spinous,  others  dark  in  colour,  "  brassy  "  ;  they  may 
also  be  altered  or  destroyed  without  being  actually  invaded 
by  the  parasite.  It  is  in  this  form  that  the  crescentic 
bodies  appear  (/,  j).  These,  however,  are  not  met  with 
at  the  very  commencement  of  the  attack,  but  appear  in 
a  week  or  so,  and  may  not  disappear  until  some  weeks 
after  the  termination  of  the  attack.  This  parasite  is  met 
with  in  the  sub-tertian,  or  so-called  malignant,  types  of 
fever,  which  are  characterised  by  irregularity  of  the  fever, 


PLATE  XXV. 


a.  Malaria.     A  tertian  "  rosette."     Smear  of  blood      X  1500. 


b.  Halteridium  DanilewsJcyi.     Smear  of  pigeon's  blood.      X  1500. 


DIAGNOSIS  OF  MALARIA  523 

considerable  blood  destruction,  often  accompanied  by 
haemoglobinuria,  and  cachexia  ;  coma  is  another  complica- 
tion in  certain  instances,  probably  caused  by  massing  of 
the  parasites  in  the  cerebral  capillaries. 

The  cure  of  malaria  by  quinine  is  regarded  as  being  due 
to  a  poisonous  action  on  the  parasites  analogous  to  that 
exerted  on  numerous  protozoa,  amoebae,  for  example, 
being  injuriously  affected  by  so  little  as  a  1-50,000  solution 
of  quinine  hydrochlorate. 

No  toxin  can  usually  be  demonstrated  in  the  blood  of 
those  suffering  from  a  malarial  attack,  but  Rosenau  and 
his  co-workers  have  found  that  the  filtered  blood,  taken 
when  the  temperature  is  rising,  produces  a  malaria-like 
paroxysm.  By  withdrawing  blood  containing  parasites, 
adding  glucose,  and  incubating  at  37°  C.,  the  multiplicative 
cycle  of  the  malaria  parasite,  as  seen  in  the  blood,  is  passed 
through  in  the  culture  tube. 

A  malaria-like  parasite  (Plas.  Kochii)  occurs  in  apes,  in  which  it 
produces  fever. 

The  nature  of  Blackwater  fever,  so  called  from  the  presence  of 
haematuria  and  haemoglobinuria,  has  given  rise  to  much  discussion. 
By  some  it  is  considered  to  be  a  disease  sui  generis,  of  unknown 
etiology.  By  others  it  is  regarded  as  a  form  of  malaria,  either  of 
an  intense  type,  or  in  which  the  kidneys  are  especially  involved,  or 
as  due  to  malarial  infection  plus  quinine.  It  may  be  that  under 
particular  conditions,  of  the  nature  of  which  we  are  at  present 
ignorant,  haemolysins  may  be  set  free  and  cause  haemolysis,  the  blood- 
pigment  being  eliminated  by  the  kidneys. 

Clinical  Examination 

The  blood  of  malarial  patients  may  be  examined  either  in  the 
unstained  or  stained  condition. 

Examination  in  the  unstained  condition. — The  finger  or  lobe  of 
the  ear  is  pricked,  and  a  droplet  of  blood  taken  up  on  a  clean  cover- 
glass,  which  is  then  placed  upon  a  slide,  so  that  the  droplet  of 
blood  spreads  out  into  a  thin  layer  between  the  two  glasses.  The 
cover-glass  may  then  be  ringed  with  oil  or  vaseline  to  prevent 


524  A  MANUAL  OF  BACTERIOLOGY 

evaporation.  A  little  practice  is  required  to  judge  the  right  quan- 
tity of  blood.  The  preparation  should  be  examined  with  a  TV-inch 
oil-immersion  lens. 

Examination  in  the  stained  condition. — To  prepare  stained  films 
the  finger  or  ear  is  pricked  of  the  malaria  or  other  blood  parasites, 
e.g.  trypanosomes,  and  a  droplet  of  blood  is  taken  up  on  the  edge 
of  the  end  of  a  slide  '(the  spreader),  which  is  then  applied  to  the 
surface  of  a  second  slide  and,  holding  the  spreader  at  an  angle  of 
45°,  it  is  pushed  along  the  surface  of  the  second  slide  so  that  a  thin 
film  of  the  blood  is  left  behind,  and  the  process  is  repeated  for  as 
many  films  as  are  required.  A  little  practice  is  required  to  gauge 
the  right  quantity  of  blood.  Other  methods  of  preparing  blood- 
films  are  to  deposit  a  droplet  of  blood  on  a  cover-glass  ;  another 
cover-glass  is  applied,  and  the  two  are  separated  so  that  each  is 
smeared  with  a  thin  film  of  blood,  or  a  droplet  of  blood  on  a  slide 
may  be  spread  with  a  cigarette  paper,  or  with  the  shaft  of  a  needle. 
Whatever  method  is  adopted,  the  film  is  allowed  to  dry  in  the 
air,  and  may  then  be  fixed  (not  if  Leishman's  stain  is  used).  In 
order  to  fix,  the  smears  should  be  immersed  in  a  mixture  of  equal 
parts  of  absolute  alcohol  and  ether  for  not  less  than  ten  minutes, 
preferably  for  half  an  hour  ;  this  gives  excellent  results.  In  hot 
countries  a  saturated  solution  of  corrosive  sublimate  may  be  used. 
The  methods  detailed  at  p.  97  may  also  be  employed. 

Staining  is  usually  carried  out  with  Leishman's  stain  (No.  12, 
p.  102).  The  blood  films,  unfixed,  are  flooded  with  a  few  drops 
(5-10)  of  the  stain,  which  is  spread  by  tilting,  and  in  hot  weather 
the  preparation  should  be  covered  with  a  capsule  to  prevent  evapo- 
ration. After  a  half  to  one  minute  distilled  water  is  added  and 
mixed  with  the  stain,  in  sufficient  amount  to  produce  an  abundant 
precipitate,  and  the  mixture  should  appear  pinkish  ;  the  water 
should  be  about  double  the  amount  of  stain  used,  and  staining  is 
continued  for  five,  or  in  some  cases  for  ten,  minutes.  The  staining 
should  be  continued  until  the  nuclei  of  the  leucocytes  are  a  rich 
purple  when  examined  with  a  low  power.  The  film  is  then  rinsed 
with  distilled  water,  a  little  distilled  water  is  left  on  the  film,  which 
is  watched  under  the  low  power  until  the  red  corpuscles  appear 
red  ;  this  takes  half  a  minute  or  more.  The  water  is  now  tilted 
off  the  film,  and  the  slide  on  edge  allowed  to  dry,  or  it  may  be  blotted 
and  dried.  Fresh  films  stain  better  than  old  ones  ;  if  the  films 
are  old,  staining  with  the  diluted  stain  should  be  prolonged  for 
ten  or  fifteen  minutes  and  differentiation  with  distilled  water 
may  take  five  minutes.  Jenner's  or  Giemsa's  blood-stain  may  be 
similarly  used. 


DIAGNOSIS  OF  MALARIA  525 

The  writer  is  indebted  to  Dr.  A.  C.  Coles  of  Bournemouth,  for 
the  following  method  of  staining  blood-parasites. 

In  order  to  obtain  good  stained  films  of  blood  containing  para- 
sites it  is  essential  to  have  good  slides,  well  cleaned,  a  film  of  blood 
spread  as  uniformly  as  possible,  and  to  avoid  any  precipitation  of 
the  stain  on  the  surface  of  the  film. 

Slides  are  best  cleaned  with  whiting  or  Creta  preparata,  made 
into  a  paste  with  water,  or  with  Windowlein,  a  preparation  used 
for  cleaning  windows.  Rub  the  whiting  thinly  over  the  surfaces 
of  the  slide,  and  when  dry  rub  off  with  a  clean  cloth. 

The  impedimenta  required  for  staining  the  blood  film  are  : 

1.  Drop  bottle  of  about  giij  capacity  containing  distilled  water  ; 

2.  Pipette  bottle  of  about  Jij  to  3iij  capacity  for  the  staining 

solution  ; 

3.  Bottle  of  Giemsa's  staining  solution  ; 

4.  Bottle  of  Merck's  pure  methylic  alcohol ;  both  well  corked  ; 

5.  A  Politzer's  bag  ;  and  preferably,  though  not  essential, 

6.  A  curved  piece  of  window  glass,  8  inch  x  4  inch. 

Into  the  perfectly  dry  pipette  bottle  pour  some  of  the  Giemsa's 
solution,  and  add  about  twice  as  much  pure  methylic  alcohol  ; 
shake  up  and  keep  well  stoppered. 

Drop  from  the  pipette  bottle  just  enough  of  the  diluted  Giemsa's 
solution  to  cover  the  film.  Allow  it  to  act  for  about  ten  to  twenty 
seconds  [if  longer,  especially  in  a  hot  climate,  the  alcohol  evaporates 
and  precipitates  the  stain]. 

Then  drop  on  as  much  distilled  water  as  the  slide  will  hold — 
that  is,  about  eight  times  as  much  water  as  stain — allow  the  stain 
and  distilled  water  to  mix,  and  stain  for  the  requisite  time. 

It  is  better,  however,  in  order  to  prevent  the  precipitation  of  the 
stain,  to  pour  off  the  diluted  stain  and  water  from  the  film  on  to 
the  surface  of  a  piece  of  slightly  curved  plate-glass,  and  immediately 
place  the  slide,  film  side  downward,  on  this.  The  duration  of 
staining  varies  according  to  the  temperature  of  the  room  and  the 
nature  of  the  film — generally  speaking,  ten  to  twenty  minutes 
give  excellent  results  ;  but  a  good  plan  is  to  remove  the  film, 
flood  off  the  stain  with  distilled  water,  and  examine  under  low 
power.  If  the  nuclei  of  the  leucocytes  are  of  a  ruby-red  colour, 
the  staining  is  successful.  If  they  are  blue,  the  film  is  insufficiently 
stained,  and  it  should  be  replaced  on  the  staining  fluid  ;  if  they 
are  blackish  red,  it  is  too  deeply  stained  for  most  purposes,  and  all 
that  is  required  is  to  pour  distilled  water  on  the  surface,  watching 
the  effect  (easily  seen  by  holding  the  slide  over  a  piece  of  white 
paper),  and  as  soon  as  the  whole  film  is  faintly  pink  the  staining 


526  A  MANUAL  OF  BACTERIOLOGY 

will  be  good.  This  method  of  staining,  generally  known  as  Giemsa's 
new  method,  closely  resembles  Leishman's,  but  very  much  more 
distilled  water  is  added. 

The  exact  tint  or  colour  of  the  objects  stained  in  this  way  will 
depend  largely  on  the  reaction  of  the  distilled  water  used  to  dilute 
the  stain.  If  the  water  is  acid  (as  most  distilled  water  is)  the 
red  blood-corpuscles  are  stained  a  reddish,  if  alkaline  they  are 
often  bluish,  in  colour. 

When  the  film  has  been  sufficiently  stained,  do  not  pour  off  the 
stain  and  then  wash,  but  flood  off  the  stain  with  distilled  water 
and  so  avoid  any  deposition  of  precipitate. 

When  the  film  has  been  quickly  washed,  it  is  essential  to  dry  it 
as  quickly  as  possible,  otherwise  decolorisation  proceeds.  The 
films  should  not  be  dried  with  filter  or  blotting-paper ;  it  tends  to 
leave  fluff.  They  are  best  dried  by  blowing  on  the  surface  with 
air  from  a  Politzer's  bag. 

Films  of  blood  which  have  been  kept  for  some  time,  especially 
in  the  tropics,  will  never  stain  well.  Films  should  therefore  be 
stained  at  once,  and  they  will  keep  indefinitely  in  a  dry  place. 
The  method  of  packing  stained  or  unstained  films  face  to  face  or 
wrapped  in  paper  is  a  barbarous  one  ;  the  surfaces  soon  get  scratched 
and  dirty.  The  best  plan  is  to  pack  them  back  to  back  in  a  racked 
box,  or  if  this  is  not  at  hand,  stick  a  small  piece  of  gummed  paper 
at  the  end  of  the  slide  on  the  film  side,  and  when  this  is  thoroughly 
dry,  but  not  before,  they  can  be  packed  together. 

It  is  essential  that  the  films  should  be  absolutely  dry  before  they 
are  mounted,  and  if  they  are  mounted  in  Canada  balsam  or  cedar-oil 
they  will  sooner  or  later  fade  and  be  perfectly  useless.  The  best  plan 
is  to  mount  them  in  parolein  or  liquid  paraffin  as  described  by  Coles 
(Lancet,  April  1,  1911),  which  has  lately  been  advocated  by  Giemsa. 

If  the  above-named  stains  are  not  available  staining  may  also 
be  done  in  a  half-saturated  aqueous  solution  of  methylene-blue  or 
in  Loffler's  blue  for  half  an  hour,  washing  in  water,  and  counter- 
staining  with  a  very  weak  eosin  solution  for  a  few  seconds,  washing 
and  drying.  Manson  recommends  treating  the  films  with  a  very 
weak  acetic  acid — two  or  three  drops  to  the  ounce  of  water — to 
dissolve  out  the  haemoglobin,  and,  after  washing,  staining  in  the 
following  solution  for  half  a  minute  : 

Borax     .......         5  parts 

Methylene-blue         .....      0-5  part 

Water 100  parts 

washing,  drying,  and  mounting  in  xylol  balsam. 


PLASMODIUM  PR^ECOX  527 

Haematoxylin  (Ehrlich's,  or  Mayer's  haemalum)  is  preferable  for 
permanent  preparations,  and  in  hot  countries,  where  methylene- 
blue  rapidly  fades.  The  preparations  may  be  counter-stained  with 
a  weak  solution  of  eosin. 

Ross  recommends  for  rapid  diagnosis  the  use  of  thick  blood  films, 
from  which  the  haemoglobin  is  first  removed  with  very  dilute  acetic 
acid  ;  the  films  are  then  stained  with  Leishman's  stain,  and 
examined  with  a  J-inch  objective.  Practice  is  required  for  this 
method. 

In  order  to  demonstrate  the  flagellated  organisms  Manson 
recommends  the  following  procedure :  Thirty  or  forty  strips  of 
thick  blotting-paper  (3  inches  by  1J  inch),  each  having  an  oblong 
hole  (^  inch  by  f  inch)  cut  lengthways  in  the  centre,  are  prepared, 
moistened  with  water,  and  laid  on  a  sheet  of  window  glass.  A 
patient  is  selected  in  whose  blood  the  crescentic  form  is  plentiful, 
and  a  minute  droplet  of  the  blood,  about  the  size  of  a  pin's  head, 
is  expressed  from  a  prick.  A  clean  slide  is  then  breathed  on,  and 
the  droplet  of  blood  picked  up  on  it  and  spread  out  with  a  needle 
so  as  to  cover  an  area  f  inch  by  £  inch.  The  slide  is  immediately 
inverted  over  a  blotting-paper  cell  and  pressed  down  sufficiently 
to  secure  perfect  apposition.  The  rest  of  the  paper  cells  are  simi- 
larly covered  with  blood-charged  slides.  In  from  half  to  three- 
quarters  of  an  hour  the  slides  are  removed  and  dried  by  gentle 
warming,  and  then  fixed  with  absolute  alcohol  for  five  minutes. 
The  alcohol  is  allowed  to  evaporate,  and  the  films  are  treated  with 
a  few  drops  of  15  per  cent,  acetic  acid  to  dissolve  out  the  haemo- 
globin. The  slides  are  then  washed  in  water  and  stained  with 
weak  carbol  fuchsin  (20  per  cent.)  for  six  to  eight  hours,  washed 
in  water,  dried,  and  mounted. 

N.B. — Negative  results  in  the  examination  for  the  malaria 
parasite  must  be  accepted  with  caution  unless  repeated.  A  single 
undoubted  parasite  is  sufficient  to  establish  the  diagnosis.  Quinine 
causes  the  disappearance  of  the  parasite.  The  parasites  in  the 
sub -tertian  fever  disappear  during  the  apyrexial  intervals  (except 
the  crescents),  and  are  most  likely  to  be  found  at  the  commencement 
of  the  attack — i.e.  when  the  temperature  is  rising.  The  parasites 
of  the  other  forms  are  larger  and  more  obvious  during  the  apyrexial 
intervals. 

[For  further  particulars  on  Malaria  and  on  the  demonstration  of 
the  malaria  parasite,  see  Daniels'  Laboratory  Studies  in  Tropical 
Medicine,  1908.] 


528  A  MANUAL  OF  BACTERIOLOGY 

Plasmodium  prsecox 

Syn.  Proteosma  Grassii,  Hcemamoeba  relicta. 

This  parasite  (commonly  called  "  proteosoma  ")  is  met  with  in 
sparrows  and  other  birds,  in  which  it  invades  the  red  blood-cor- 
puscles, and  its  structure  and  development  are  practically  identical 
with  those  of  the  benign  malarial  parasites  of  man.  It  grows  from 
a  minute  granule  into  an  amoeboid  plasmodium,  which  ultimately 
segments  and  forms  a  rosette.  In  some  specimens  of  blood  flagel- 
lated male  gametocytes  make  their  appearance,  similar  to  those 
of  malaria,  the  flagella  break  away  from  the  main  mass,  fertilise 
other  non-flagellated  or  female  cells,  and  a  series  of  changes  ensues 
analogous  to  those  occurring  in  the  malaria  parasite  (p.  516).  The 
fertilisation  and  development  of  the  fertilised  cell  take  place  in  the 
stomach  of  a  mosquito  (Culex  fatigans),  by  which  the  infection  is 
transmitted  to  other  birds. 

Halteridium  Danilewskyi 

This  is  an  elongated,  curved  parasite  (also  known  as  Hcemo- 
proteus  or  Hcemamoeba  Danilewskyi},  found  in  the  red  corpuscles  of 
certain  birds  (pigeon,  crow,  etc.),  and  embracing  the  nucleus  (Plate 
XXV.  b).  By  some  it  is  included  among  the  malaria-like  parasites 
(Plasmodium).  At  an  early  stage  it  much  resembles  the  proteosoma, 
but  as  it  grows  it  becomes  elongated,  pigment -granules  appear, 
and  are  either  scattered  throughout  the  protoplasm  or  collect  in 
two  groups,  one  at  each  extremity.  Finally,  the  parasite  occupies 
nearly  the  whole  of  the  corpuscle,  dislocating  its  nucleus.  The 
fully  grown  parasites  may  be  differentiated  into  two  forms,  one 
of  which  remains  almost  completely  unstained  when  treated  with 
methylene-blue,  the  other  staining  deeply  with  this  dye  (Opie). 
When  the  blood  is  withdrawn,  the  corpuscles  disintegrate  and 
liberate  the  contained  parasites,  which  assume  a  circular  outline, 
and  a  certain  number  become  flagellated.  It  is  only  the  non-staining 
form  which  becomes  flagellated.  These  two  varieties  of  the  parasite 
are  the  male  and  female  cells  respectively,  and  the  fertilisation  of 
the  female  cell  by  a  free  flagellum  has  been  actually  observed  by 
MacCallum.1  It  can  hardly  be  doubted  that  the  development  of 
the  fertilised  cells  takes  place  in  some  insect,  but  the  definitive 
host  has  not  yet  been  discovered  with  certainty. 

The  presence  of  these  parasites  induces  rise  of  temperature, 

1  Journ.  Exper.  Mecl,  vol.  iii,  1898,  pp.  79,  103,  117. 


THE  PIROPLASMATA  529 

deposition  of  melanin,  and  changes  in,  and  enlargement  of,  the 
spleen  and  liver,  analogous  to  those  occurring  in  malaria  in  man. 
The  Halteridium  parasite,  according  to  Schaudinn,  is  a  stage  in 
the  life-cycle  of  a  trypanosome  (see  p.  494). 

Somewhat  similar  parasites  are  frequent  in  the  blood  of  the  lower 
vertebrates  (see  Plate  XXVI.  &). 


The  Piroplasmata  x 

Syn.  Pyrosoma,  Bdbesia. 

The  Piroplasmata  form  a  somewhat  anomalous  group,  but  are 
usually  included  in  the  Haemosporidia  of  the  Sporozoa.  They 
differ  from  the  Plasmodia  in  the  following  respects  :  absence  of 
pigment,  non-fragmenting  of  the  nucleolus,  division  into  two  or 
four  only,  and  frequency  of  extra-corpuscular  forms.  They  cause 
many  diseases  in  animals,  are  conveyed  by  ticks,  but  are  unknown 
in  man.  (A  piroplasma  was  described  as  the  causative  organism 
of  Rocky  Mountain  spotted  fever  by  Wilson  and  Chowning,  but 
the  observations  appear  to  be  erroneous,  see  p.  546).  The  body 
of  a  piroplasma  is  typically  pear-shaped  (Plate  XXVI.  a),  but 
rounded  and  rod  forms  occur.  Two  nuclear  masses  are  present, 
one  larger  than  the  other. 

The  developmental  cycle  in  the  ticks  has  not  been  worked  out, 
but  Koch  has  observed  peculiar  rayed  forms  with  P.  bigeminum, 
and  Christophers  2  various  developmental  forms  with  P.  canis. 
Miyajima  states  that  a  piroplasma  of  Japanese  cattle  (apparently 
P.  parvum)  in  blood  broth  develops  into  typical  trypanosome  forms.3 

Piroplasma  bigeminum. — This  is  the  parasite  of  the  well-known 
Texas  fever  of  cattle,  a  disease  which  is  characterised  by  fever, 
emaciation,  anaemia,  haemoglobinuria,  and  enlargement  of  the  liver 
and  spleen. 

The  disease  causes  considerable  loss  among  cattle,  and  is  met 
with  in  various  parts  of  the  world,  America,  Australia,  South  Africa, 
Malaya,  the  Philippines,  the  Roman  Campagna,  Greece,  Roumania, 
and  North  Ireland. 

In  the  acute  type  of  the  disease  a  small  proportion  (1-5  per  cent.) 
of  the  red  corpuscles  in  the  peripheral  circulation  contain  pairs  of 

1  See   Hewlett,   Trans.   XlVtk  Internal.   Cong,   of    Hygiene,   Berlin, 
vol.  ii,  1908,  p.  146  ;   Minchin  in  Allbutt's  System  of  Med.,  ed  2,  vol.  ii, 
pt.  2,  p.  86. 

2  Brit.  Med.  Journ..  1907,  vol.  i,  p.  76. 

3  Philippine  Journ.  of  Science,  vol.  ii,  1908,  p.  37. 

34 


530  A  MANUAL  OF  BACTERIOLOGY 

pyriform  bodies  2-4  /j.  in  length  and  1-5-2  p.  in  largest  diameter. 
One  end  of  each  body  is  rounded,  and  the  body  gradually  tapers  to 
a  point  at  the  other  end,  and  the  pair  lie  close  together,  their  tapering 
ends  directed  towards  each  other.  A  dark  spherical  body  is  present 
at  the  rounded  end  of  the  parasite. 

Some  of  the  young  parasites  exhibit  amoeboid  movements  when 
the  blood  is  examined  on  a  warm  stage.  In  the  internal  organs 
the  parasites  are  more  numerous  ;  in  the  kidney  and  liver  10-25 
per  cent,  of  the  corpuscles  contain  them,  in  the  heart-muscle 
50  per  cent.  In  the  mild  type  5-50  per  cent,  of  the  corpuscles  in 
the  circulating  blood  may  be  infected  at  one  time  or  another,  and 
the  parasite  appears  in  some  cases  as  a  coccus-like  body  at  the 
periphery  of  the  corpuscle.  This  appears  to  become  enlarged  and 
spindle-shaped,  then  to  taper  in  the  middle,  divide,  and  so  give  rise, 
to  the  pyriform  bodies.  Occasionally  minute  free  coccoid  bodies 
are  seen  in  the  plasma,  and  at  times  two  to  five  minute  (0-5  p) 
coccoid  cells  are  present  in  the  red  cells.  After  death  the  pyriform 
bodies  seem  to  become  spherical  or  angular. 

Sexually  differentiated  gametes  are  not  known  with  certainty 
but  flagellated  forms  have  been  described. 

The  disease  is  transmitted  through  the  bites  of  ticks  (Rhipi- 
cephalus  annulatus,  R.  australis).  The  female  tick,  after  biting  an 
infected  ox  and  sucking  its  blood,  falls  off  and  lays  its  eggs  ;  the 
eggs  hatch  in  two  to  six  weeks'  time,  and  the  daughter  ticks  transmit 
the  disease  to  other  animals  through  their  bites.1  The  disease  may 
be  to  some  extent  controlled  by  prophylactic  measures  designed 
to  destroy  the  ticks,  and  to  prevent  infection  thereby. 

A  partial  immunity  is  enjoyed  after  an  attack  of  the  disease, 
but  by  repeated  attacks  the  immunity  may  be  rendered  absolute. 
By  inoculation  with  the  blood  of  an  affected  animal  in  which  the 
fever  has  subsided,  a  transient  illness  in  the  inoculated  animal 
is  produced  together  with  partial  immunity,  and  by  a  second 
or  third  inoculation  the  immunity  may  be  much  increased.  The 
mortality  from  such  a  procedure  amounts  to  3-5  per  cent.2 

P.  parvum  causes  Rhodesian  red-water  of  cattle.  It  is  not 
directly  inoculable,  and  is  conveyed  by  the  tick  R.  appendiculatus. 

P.  equi  causes  biliary  fever  in  horses. 

P.  canis  causes  epidemic  jaundice  in  dogs  (Plate  XXVI.  a). 
It  is  conveyed  by  the  ticks  Hcemaphysalis  leachi  in  South  Africa, 

1  See  Smith  and   Kilborne,  Texas  or  Southern  Cattle  Fever,  United 
States  Dep.  Agricult.  Bull.  No.  1,  1893. 

2  See  Tidswell,  Report  on  Protective  Inoculation  against  Tick  Fever, 
New  South  Wales,  Dep.  Pub.  Health,  vol.  i,  1898  ;  vol.  ii,  1900. 


PLATE  XXVI. 


a.  Piroplasma  canis.     Film  of  blood,      x  1500. 


&.  Hwmocystidium  (Hcemoproteus)  najce.     Pigmented  parasite  of 
Cobra  (Naja  hajce). 


MICROSPORIDIA  531 

E.  sanguineus  in  India,  and  Dermacentor  reticulatus  in  Europe.1 
(On  Ticks,  see  Nuttall,  Journ.  Eoy.  Inst.  of  Public  Health,  vol.  xvi, 
1908,  p.  385.) 

H  aemogregarina 

The  Hsemogregarines  (which  must  be  distinguished  from  the 
Gregarines)  are  unpigmented  parasites,  not  amoeboid,  typically 
having  an  elongated  body  or  vermicule,  occurring  in  the  blood, 
mostly  in  cold-blooded  vertebrates,  but  several  species  have  of 
late  been  found  in  mammals  (dog,  jerboa,  palm  squirrel),  though 
not  in  man.  In  the  dog,  the  parasite  (Leucocytozoon  canis)  occurs 
as  an  elongated,  curved  or  doubled-up  body  in  the  polymorphonu- 
clear  leucocytes.  It  is  encapsuled  and  contains  a  single  granular 
nucleus.  Encystment  with  sporulation  occurs  in  the  bone -marrow, 
and  a  sexual  development  is  stated  to  occur  in  a  tick. 

Hcemogregarina  (Drepanidium,  Lankesterella)  ranarum  inhabits 
frogs  (Eana  esculenta),  and  possesses  both  an  intra-  and  an  extra- 
corpuscular  phase.  In  the  former  the  parasite  occurs  as  an  elon- 
gated gregarine-like  body  within  the  red  corpuscles,  which  increases 
in  size  until  its  length  is  10-15  p  ;  it  then  divides  into  numerous 
small  or  a  few  large  gymnospores.  In  the  first  case  the  spores 
may  number  fifty,  are  3-5  p  in  length,  occur  in  May  or  June,  and 
are  exclusively  within  the  erythrocytes  ;  in  the  latter  case  the 
spores  measure  5-8  \i  in  length,  are  five  to  fifteen  in  number,  and 
develop  within  cells  in  the  blood-forming  organs.  The  extra- 
corpuscular  phase,  commencing  within  the  corpuscles,  ends  in  an 
elongated  organism  possessing  a  vermicular  movement,  and  free 
in  the  plasma.  Similar  parasites  are  frequent  in  the  lower  verte- 
brates, e.g.  snakes. 

Order. — Myxosporidia 

In  this  group  the  trophozoite  is  amoeboid,  and  the  species  are 
almost  exclusively  parasites  of  fish,  in  the  young  stage  being  intra- 
cellular  ("  fish  psorosperms  "). 


Order. — Microsporidia 

The  Microsporidia  are  cell  parasites  of  invertebrates,  especially 
arthropods,  and  the  trophozoite  is  more  or  less  amoeboid. 

1  See  Nultall  and  Grab  am -Smith,  Journ.  of  Hygiene,  vols,  iv  to  viji, 
1904-8. 


532  A  MANUAL  OF  BACTERIOLOGY 

Nosema  bombycis  causes  pebrine,  a  disease  of  silkworms,  which 
is  of  considerable  importance  commercially,  for  the  silk  industry 
in  France  was  once  threatened  with  extinction  owing  to  its  ravages. 
The  infected  worms  do  not  grow  normally,  cease  to  eat,  and  die,  or 
may  form  abnormal  pupse.  Within  the  body  of  the  affected  worms 
a  large  number  of  roundish,  highly  refractile  corpuscles  are  found. 
Pasteur  ascertained  that  the  disease  was  propagated  by  healthy 
worms  eating  with  their  food  the  excreta  of  infected  ones.  The 
moths  were  thus  infected,  and  laid  infected  eggs.  By  allowing  each 
moth  to  lay  its  eggs  separately,  and  subsequently  examining  the 
body  of  the  moth  microscopically,  he  was  able  to  separate  the  healthy 
from  the  diseased,  and  the  eggs  of  the  former  were  kept,  while  those 
of  the  latter  were  destroyed.  According  to  Pfeiffer,1  when  the 
worms  eat  the  excreta  containing  the  corpuscles  mentioned  above, 
these  lose  their  capsule  and  form  large  amoeboid  masses  which 
penetrate  the  muscles  and  blood-corpuscles.  The  amoeboid  masses 
then  become  encapsuled  and  are  yellow  and  granular.  Later  on 
the  bright  roundish  corpuscles  form  within  them. 

The  Isle  of  Wight  bee  disease  is  caused  by  Nosema  apis,  which 
is  mainly  confined  to  the  alimentary  tract. 

Another  disease  of  silkworms  is  known  as  flacherie,  but  is  due 
to  a  bacterium,  Micrococcus  bombycis.  It  is  contagious,  and  can 
be  transmitted  by  inoculation. 


Order. — Sarcosporidia 

The  parasites  belonging  to  this  order  are  not  thoroughly  worked 
out.  They  complete  their  life-history  in  the  substance  of  striated 
muscular  fibres :  such  are  the  well-known  Miescher's  corpuscles. 
Few  instances  of  this  class  of  parasite  are  recorded  in  man,  but  it 
occurs  in  the  monkey  2  and  also  in  the  ox.  T.  Smith  3  describes 
the  characters  and  development  of  a  species  found  in  mice. 

A  parasite,  Rhinosporidium  kinealyi,  nearly  allied  to  the  fore- 
going, causes  a  polypoid  condition  in  the  nose  in  the  tropics.  If 
a  section  be  made  of  the  mass,  cysts  (pansporoblasts)  will  be  seen 
in  the  deeper  layers  containing  many  refractile  rounded  nucleated 
bodies,  the  spores.  Neither  the  life-history  nor  the  mode  of  trans- 
mission of  the  parasite  is  known. 

1  Zeitschr.f.  Hyg.,  vol.  iii,  1888,  p.  3. 

2  De  Korte,  Journ.  of  Hygiene,  vol.  v,  1905,  p.  451 

3  Journ.  Exper.  Med.,  vol.  vi,  No.  1,  1901,  p.  1. 


CHAPTER  XIX 

SCARLET  FEVER— HYDROPHOBIA— INFANTILE  PARA- 
LYSIS  —  TYPHUS  FEVER  —  YELLOW  FEVER  - 
DENGUE— PHLEBOTOMUS  FEVER— VACCINIA  AND 
VARIOLA— MALIGNANT  DISEASE 

Scarlet  Fever 

VARIOUS  organisms  have  been  described  in  scarlet  fever — 
a  bacillus  by  Eddington,  a  streptococcus  by  Frankel  and 
Freudenberg,  protozoa  by  Mallory  and  others.  The 
disease  may  be  milk-borne,  and  in  the  historic  Hendon 
outbreak  a  streptococcus  was  claimed  by  Klein  to  be 
the  specific  infective  agent,  but  the  researches  of  Crookshank 
and  others  seem  to  disprove  this. 

In  1885  an  epidemic  of  scarlet  fever  occurred  in  Mary- 
lebone,  and  was  traced  to  infection  conveyed  by  milk 
supplied  from  a  farm  at  Hendon.  The  infection  could 
not  be  traced  to  any  human  source,  and  it  was  therefore 
concluded  that  the  cows  themselves  were  affected  with 
scarlet  fever,  and  infected  the  milk.  A  vesicular  eruption 
was  found  on  the  udders  and  teats  of  the  cows,  and  this 
was  regarded  as  the  local  manifestation  of  bovine  scar- 
latina. From  the  vesicles  and  crusts  Klein  isolated  a 
streptococcus  which,  although  closely  resembling  the 
Streptococcus  pyogenes  (as  then  known),  differed  slightly 
from  it ;  on  inoculation  into  calves  it  produced  death, 
with  lesions  of  the  kidney  resembling  those  of  the  human 
disease.  Klein  also  isolated  the  same  streptococcus  in 

533 


534  A  MANUAL  OF  BACTERIOLOGY 

five  out  of  eleven  cases  of  the  disease  in  man.  The  con- 
clusions which  Klein  and  Power  came  to  were,  therefore, 
that  scarlet  fever  is  communicable  to,  and  may  exist  in 
cows,  the  milk  thereby  becoming  infected  and  conveying 
the  disease  to  man,  and  that  a  streptococcus  is  the  specific 
infective  agent. 

The  Hendon  outbreak  was  reinvestigated  by  Axe  and 
Crookshank.1  Axe  found  that,  so  far  from  there  being 
no  source  of  human  infection,  cases  of  scarlet  fever  had 
occurred  near  the  dairy  within  a  short  time  of  the  out- 
break, and  the  eruptive  disease  of  the  cow  was  shown  by 
Crookshank  to  be  cowpox,  while  the  so-called  streptococcus 
of  scarlet  fever  he  regarded  as  a  variety  of  the  S.  pyogenes. 
The  existence  of  bovine  scarlet  fever  is  entirely  discredited 
by  the  veterinary  profession,  both  here  and  on  the  Continent. 

In  1909  a  milk-borne  epidemic  occurred  in  certain 
districts  in  London  and  Surrey,  and  was  traced  to  milk 
derived  from  one  farm.  The  outbreak  was  investigated 
and  reported  on  by  Hamer  and  Jones,  who  again  traced 
it  to  infection  of  the  cows.  Hunting  2  reviews  the  evidence 
and  shows  how  little  there  is  to  support  this  conclusion, 
as  there  is  no  doubt  that  the  family  of  one  of  the  employees 
on  the  farm  were  suffering  from  scarlatina. 

Scarlatina  seems  to  be  inoculable  in  the  chimpanzee 
and  some  of  the  lower  apes.  It  is  now  regarded  as  being 
caused  by  a  filter-passer. 

Gordon 3  reinvestigated  the  bacteriology  of  scarlatina  with 
special  reference  to  the  Streptococcus  scarlatince  or  conglomeratus 
of  Klein.  He  found  that  this  organism  differs  distinctly  in  its 
cultural  characters  from  other  varieties  of  streptococci,  and  that 
it  occurs  constantly  in  the  mucous  secretion  on  the  surface  of  the 
tonsils  and  fauces  and  in  the  nasal,  but  not  in  the  aural,  discharge 

1  On  the  Hendon  outbreak,  see  Trans.  Path.  Soc.  Lond.,  1888  (Refs.). 

2  Journ.  Roy.  Sanitary  Inst.,  vol.  xxxii,  1911,  p.  62. 

3  (a)  Rep.  Med.  Off.  LOG.  Gov.  Board  for  1898-99,  p.  480  ;     (b)  ibid. 
for  1899-1900,  p.  385. 


HYDROPHOBIA  535 

in  scarlatina.  It  is  also  present  in  a  somewhat  modified  form  in 
the  blood  and  tissues  post  mortem.  It  was  not  found  in  four 
non-scarlatinal  throats  examined.  Gordon  concluded,  therefore, 
that  the  S.  scarlatina  or  conglomeratus  is  the  "specialised  and 
essential  agent  "  of  scarlatina.  It  is  pathogenic  to  mice. 

Cumpston  !  investigated  the  biological  characters  of  101  strep- 
tococci isolated  from  scarlet  fever,  applying  Gordon's  tests  (p.  233). 
The  majority  corresponded  with  the  S.  longus  type. 

Baginsky  and  Sommerfeld,  Class  and  Jaques  also  isolated  strep- 
tococcoid  organisms  in  scarlatina,  but  they  possessed  no  very 
distinctive  cultural  characters. 

It  seems  very  doubtful  if  streptococci  are  the  etiological  agents 
in  scarlet  fever ;  they  are  probably  secondary  infective  agents. 
It  is  remarkable  how  frequently  diphtheria  complicates  scarlatina. 

Mallory  detected  small  bodies,  2-7  p  in  diameter,  staining  deli- 
cately but  sharply  with  met hylene -blue,  and  occurring  in  and 
between  the  epithelial  cells  of  the  epidermis  and  in  the  lymph- 
vessels  and  spaces  of  the  corium.  He  regards  these  as  protozoa, 
but  others  consider  them  to  be  degenerated  leucocytes  (see  p.  537). 

The  blood  in  the  early  stages  of  scarlatina  gives  the  Wassermann 
reaction  (p.  502). 

% 

Hydrophobia  2 

Hydrophobia  attacking  man  is  invariably  contracted 
through  the  bite  of  an  animal  affected  with  the  disease, 
In  the  lower  animals  the  disease  is  termed  rabies,  and  is 
most  frequent  in  the  dog,  but  the  cat,  wolf,  and  deer  are 
also  subject  to  it,  and  other  animals  can  be  infected  by 
inoculation.  The  disease  may  assume  two  forms — the 
raging  and  the  paralytic.  The  latter  is  not  met  with  in 
man,  unless  certain  rare  forms  of  acute  ascending  paralysis 
(e.g.  Landry's)  be  manifestations  of  it.  In  the  dog  either 
may  occur,  but  in  rodents  the  paralytic  form  is  almost 
always  the  one  assumed.  In  man  the  incubation  period 
is  very  variable  ;  it  is  never  less  than  about  twenty  days, 

1  Journ.  of  Hyg.,  vol.  vii,  1907,  p.  599. 

2  See  Marie,  La  Rage,  1901  ;   Scientific  Memoirs  Gov.  of  India,  Nos. 
30  and  44;     Luzzani,  Ann.  de  VInst.  Pasteur,  xxvii,   1913,  p.   1039 
(Bibliog.). 


536  A  MANUAL  OF  BACTERIOLOGY 

and  possibly  may  be  as  long  as  two  years,  or  even  more ; 
the  average  seems  to  be  about  ten  weeks.  In  the  rabbit, 
after  inoculation  from  the  dog,  the  incubation  period  is 
about  two  to  three  weeks. 

The  virus  resides  in  the  central  nervous  system,  as  was 
shown  by  Pasteur.  Inoculation  with  emulsions  prepared 
from  the  medulla  and  with  the  saliva  conveys  the  disease, 
but  the  filtered  emulsions  are  usually  inactive,  and  the 
other  tissues  and  fluids  of  the  body,  excepting  the  lacrimals 
and  suprarenals,  are  non-infective. 

Remlinger  *  has  found  that  after  very  complete  tritura- 
tion  the  virus  may  pass  through  a  porcelain  filter. 

No  micro-organism  has  been  demonstrated  with  certainty 
in  rabies.  Negri  has  described  the  constant  presence  of 
structures — the  Negri  bodies — particularly  in  the  grey 
matter  of  the  hippocampus  major,  which  he  regards  as 
protozoa.  They  are  of  varying  size,  apparently  encap- 
suled,  taking  a  homogeneous  purplish  colour  in  smears 
stained  with  eosin  and  methylene-blue,  the  smallest 
spherical  and  structureless,  larger  ones  with  a  central 
granule  or  nucleus,  the  largest,  round,  ovoid  or  elongated, 
containing  several  (as  many  as  eight)  granules  (Fig.  65). 
They  occur  abundantly  in  animals  suffering  from  chronic 
rabies,  but  in  the  acute  type  are  scanty,  though  still  to 
be  found  ;  in  "  fixed  virus  "  (p.  538)  they  are  very  small. 
So  constantly  are  the  Negri  bodies  present  in  rabies,  and 
absent  in  non-rabic  conditions,  that  their  presence  or 
absence  forms  a  rapid  and  simple  means  of  diagnosis.2 

Inasmuch  as  the  rabies  virus  is  filterable,  the  view 
taken  by  Prowazek  of  the  nature  of  the  Negri  bodies  is 
that  they  represent  the  tissue  reaction  to  invasion  by  the 
parasite,  the  parasite  being  an  extremely  minute  one 

1  Bull,  de  I'InsL  Pasteur,  iv,  1904,  p.  342. 

2  See  Williams  and  Lowden,  Journ.   Infect.  Diseases,  vol.  iii,  1906, 
p.  452. 


HYDROPHOBIA 


537 


and  contained  within  the  body  and  belonging  to  a  group 
of  the  Protozoa  termed  the  Chlamydozoa.  In  the  same 
category  he  would  place  the  trachoma  bodies,  the  Mallory 
bodies  of  scarlatina  and  the  Councilman  bodies  of  variola. 
Noguchi  believes  that  the  Negri  bodies  or  derivatives 
from  them  can  be  cultivated  in  his  medium  used  for  the 
Trep.  pallidum  (p.  497). 

Babes  states  that  the  virus  is  destroyed  at  a  tempera- 
ture of  60°  C.,  but  the  medulla  and  other  infective  material 


FIG.  65. — Smear  from  hippocampus  major  of  rabid  dog : 
n,  nucleus  of  nerve-cell;  b.  b,  the  Negri  bodies  (eosin 
and  methylene-blue).  (After  Williams  and  Lowden.) 

retain  their  virulence  for  months  in  glycerin.  He  has 
described  certain  lesions  present  in  the  medulla  in  cases 
of  rabies,  the  so-called  rabic  tubercles.  These  consist  of 
an  invasion  of  the  peri-ganglionic  spaces  by  an  accumulation 
of  round-cells,  with  degeneration  of  the  cells  of  the  bulbar 
nuclei. 

Van  Gehuchten  has  described  as  pathognomonic  of 
rabies  certain  lesions  in  the  sympathetic  and  cerebro- 
spinal  ganglia,  especially  those  of  the  pneumo-gastric. 
These  ganglia  consist  normally  of  a  supporting  tissue 


538  A  MANUAL  OF  BACTERIOLOGY 

holding  in  its  meshes  large  ganglionic  cells  with  distinct 
well-staining  nuclei,  each  being  enclosed  in  a  capsule 
lined  with  endothelium.  The  changes  in  rabies  consist 
in  atrophy  of  the  ganglionic  cells,  which  become  shrunken 
and  no  longer  fill  the  enclosing  capsule,  and  their  nuclei 
at  the  same  time  become  ill-defined  and  stain  badly. 
A  number  of  new-formed  cells  also  appear  within  the 
ganglionic  capsules.  Ravenel  and  McCarthy  studied 
twenty- eight  cases  of  rabies  in  various  animals,  and  consider 
that  these  capsular  and  cellular  changes  in  the  ganglia, 
taken  in  conjunction  with  the  clinical  manifestations, 
afford  a  rapid  and  trustworthy  means  of  diagnosis  of 
rabies,  but  that  the  absence  of  these  changes  does  not 
necessarily  imply  that  rabies  is  not  present.  They  also 
consider  that  the  rabic  tubercle  of  Babes  is  present  suffi- 
ciently often  to  furnish  valuable  assistance  in  cases  where 
the  central  nervous  system  only  is  obtainable.1 

Pasteur  showed  that  the  virus  can  be  attenuated  by 
desiccating  the  infective  nerve  matter,  and  in  this  way 
was  able  to  prepare  a  vaccine  which  protects  animals  from 
otherwise  fatal  doses  of  the  virus.  Advancing  a  step 
further,  he  used  his  vaccines  to  treat  individuals  who  had 
been  bitten  by  rabid  animals,  but  in  whom  the  symptoms 
had  not  yet  developed,  and  so  inaugurated  the  present 
system  of  anti-rabic  inoculation  as  carried  out  at  the 
Pasteur  and  other  institutes. 

To  prepare  the  anti-rabic  vaccines,  a  rabbit  is  inocu- 
lated subdurally  with  an  emulsion  made  from  the  medulla 
of  a  rabid  dog.  When  the  animal  dies,  a  second  rabbit  is 
similarly  inoculated  from  the  first,  and  the  passage  through 
rabbits  is  continued  until  a  "  fixed  "  virus  is  obtained, 
with  which  the  first  symptoms  appear  on  the  seventh  or 
eighth  day,  and  which  kills  with  certainty  in  about  ten 

1  See  Journ.  Compar.  Pathol.  and  Therapeut.,  vol.  xiv,  pt.  i,  1901, 
p.  37. 


HYDKOPHOBIA  539 

days.  This  having  been  attained,  two  or  three  rabbits 
are  inoculated  subdurally  every  day,  so  that  there  is  a 
daily  supply  of  animals  dead  of  the  disease.  The  spinal 
cord  is  removed  with  aseptic  precautions,  cut  into  con- 
venient segments,  and  suspended  in  bell  jars  containing 
a  layer  of  caustic  potash  at  the  bottom,  which  serves  to 
desiccate  them.  The  jars  are  dated,  and  preserved  in 
glass  cases  in  a  dark  room,  kept  at  a  constant  temperature 
of  about  23°  C.  In  Paris  the  vaccine  fluids  are  prepared  by 
triturating  portions  of  the  dried  cords  in  sterile  broth, 
so  as  to  form  an  emulsion — 1  cm.  of  cord  in  5  c.c.  of  sterile 
broth,  of  which  1  c.c.  (i.e.  2  mm.  of  cord)  forms  a  single 
dose.  At  the  commencement  of  treatment  the  cords 
which  have  been  dried  for  fourteen  days  are  used,  at  the 
end  of  treatment  those  which  have  been  dried  for  only 
three  days  ;  the  latter  are  much  more  virulent,  and  would 
communicate  the  disease  but  for  the  previous  treatment. 
The  rabbits  employed  should  all  be  of  the  same  weight 
(2J  kilogrammes  in  Paris) ;  if  the  rabbits  are  small,  a 
slightly  shorter  period  of  desiccation  of  the  cords  would 
be  necessary.  The  treatment  varies  in  duration  according 
to  the  severity  of  the  case,  which  is  gauged  by  the  number 
and  situation  of  the  bites  and  by  the  species  of  animal. 
Bites  on  exposed  parts  are  regarded  as  much  more  serious 
than  those  through  clothing,  and  on  the  face,  where 
efficient  treatment  is  difficult,  than  on  the  hands,  and 
wolf-bites  than  dog-bites. 

The  doses  are  injected  subcutaneously  in  the  flank, 
and  do  not  produce  much  constitutional  disturbance. 
At  first  there  is  a  feeling  of  lassitude,  and  considerable 
muscular  tenderness  at  the  seat  of  inoculation,  which 
later  on  passes  off.  At  Lille,  where  there  are  only  a  few 
cases  under  treatment  at  a  time,  the  cords,  after  drying 
for  the  requisite  period,  are  placed  in  pure  sterile  glycerin. 
In  this  they  retain  their  virulence  unimpaired  for  about 


540 


A  MANUAL  OF  BACTERIOLOGY 


a  month.  This  method  does  away  with  the  necessity  for 
the  daily  inoculation  of  rabbits,  a  rabbit  being  inoculated 
occasionally  as  required.  The  system  of  dosage  employed 
at  the  various  anti-rabic  stations  differs  somewhat ;  the 
following  is  that  employed  at  Lille,  2  mm.  of  cord  being 
emulsified  in  5  c.c.  of  sterile  broth,  or  physiological  salt 
solution  : 


ORDINARY  TREATMENT. 


ORDINARY  TREATMENT. 


Day  of                                         Days  of 

Day  of                                         Days  of 

treat-                                       desiccation 

treat-                                        desiccation 

ment.                                          of  cord. 

ment.                                         of  cord. 

1  (two  injections) 

14  and  13 

13     .          .        . 

3 

2 

12  and  11 

14  (two  injections) 

9  and  8 

3 

10  and  9 

15 

7  and  6 

4 

8  and  7 

16  . 

5 

5  . 

6 

17  . 

4 

6  . 

5 

18  . 

3 

7 

4 

8  .      !      ! 

3 

9  (two  injections) 

9  and  8 

FOR  SEVERE  BITES,  in  Addition. 

10 

7  and  6 

19  (two  injections)     .        7  and  6 

11   . 

5 

20                 .,               .        5  and  4 

12   . 

4 

21   .         .         .         .3 

At  Buda-Pesth  a  dilution  method  has  been  employed ; 
instead  of  drying  the  cords,  an  emulsion  is  made  with 
the  fresh  cord,  and  this  emulsion  is  considerably  diluted 
for  the  earlier  doses,  dilutions  of  1  in  10,000  to  1  in  6000, 
corresponding  to  cords  dried  for  from  fourteen  to  eight 
days.  Semple  *  has  found  that  a  carbolised  emulsion 
of  the  cord  may  be  employed  as  the  inoculating  agent. 
An  8  per  cent,  emulsion  of  the  cord  in  physiological  salt 
solution  with  1  per  cent,  carbolic  acid  is  kept  at  37°  C. 
for  twenty-four  hours.  At  the  end  of  this  time  an  equal 
volume  of  physiological  salt  solution  is  added  and  the 
emulsion  bottled  aseptically.  This  vaccine  will  keep  for 
months. 

Undoubtedly  the  Pasteur  inoculations  will  protect 
animals  from  rabies,  the  duration  of  immunity  after 

1  Sc.  Mem.  Gov.  of  India,  No.  44. 


ANTI-KABIC  INOCULATION  541 

vaccination  in  the  dog  being  at  least  three  years.  In  man 
the  efficacy  of  the  treatment  can  only  be  judged  by 
statistics.  The  mortality  after  bites  by  supposed  rabid 
animals  is  variously  stated,  the  most  favourable  being 
about  16  per  cent.  (Leblanc).  At  the  Pasteur  Institute, 
Paris,  among  2730  cases  treated  in  which  the  animal 
which  inflicted  the  bites  was  proved  to  be  rabid  by  inocu- 
lation experiments,  nineteen  deaths  occurred — a  mortality 
of  O7  per  cent.  In  1910,  401  cases  were  treated,  with 
no  death ;  in  1911,  341  cases,  with  one  death ;  in  1912, 
395  cases,  with  no  death,  being  mortalities  of  O'OO,  0'29, 
and  0*00  per  cent,  respectively. 

The  failure  of  the  treatment  may  be  due  to  two  causes  : 
(1)  delay  in  its  commencement,  and  (2)  a  short  incubation 
period.  The  principle  of  the  treatment  probably  depends 
upon  the  long  incubation  period  of  the  disease,  owing  to 
which  it  is  possible  to  forestall  the  disease,  and  to  immunise 
the  body  by  the  inoculations  before  its  onset.  If,  unfor- 
tunately, the  infective  material  should  be  very  virulent, 
and  the  incubation  period  thereby  reduced  to  the  lower 
limit,  it  may  be  impossible  to  do  this  before  the  onset 
of  the  disease,  and  the  same  is  the  case  if  the  commence- 
ment of  the  treatment  be  delayed.  Pasteur's  system  of 
inoculation  is  useless  when  the  disease  has  declared  itself. 

By  vaccinating  animals  by  the  Pasteur  method  by  a 
long  series  of  injections,  and  with  the  most  virulent  material, 
the  blood-serum  acquires  "  anti-  "  properties,  and  this 
"  anti-rabic  "  serum  is  said  to  be  of  service  in  the  treatment 
of  the  declared  disease. 

Variations  from  typical  rabies  have  been  described  both  in 
animals  and  in  man  under  such  names  as  "  chronic  rabies,"  "  abor- 
tive rabies,"  etc.  Harvey,  Carter,  and  Acton  *  describe  a  spon- 
taneous disease  in  dogs  due  to  a  general  infection  with  B.  pyocyaneus. 
which  closely  simulates  rabies.  By  subdural  inoculation  the  disease 

1  Veterinary  Record,  July  22,  1911,  p.  57. 


542  A  MANUAL  OF  BACTERIOLOGY 

is  reproduced  in  rabbits,  with  paresis  of  the  hind  legs  and  death  in 
from  sixteen  to  twenty-one  days.  The  Negri  bodies  are  absent, 
the  course  of  the  disease  differs  somewhat  from  rabies,  and  the 
B.  pyocyaneus  can  be  isolated  from  the  brain  and  blood. 


Diagnosis  of  Rabies 

In  a  case  of  suspected  rabies  in  a  dog  the  animal  should  not  be 
killed  immediately,  but  should  be  kept  under  observation  until 
it  dies,  or  for  three  or  four  weeks,  and  then  killed. 

1.  Moderately  thin  smears  on  slides  are  made  from  (a)  the  cortex 
in  the  region  of  the  fissure  of  Eolando  (the  crucial  sulcus  in  the 
dog),  (b)  the  hippocampus  major,  (c)  the  cerebellum.  They  are 
dried  in  the  air,  fixed  for  five  minutes  in  methyl  alcohol,  and  then 
stained  in  weak  Giemsa  (1  drop  stain,  1  c.c.  distilled  water  ;  with 
1  drop  of  1  per  cent,  potassium  carbonate  solution  to  every  10  c.c. 
of  the  dilute  stain)  for  three  hours.  The  stained  films  are  then 
washed  in  running  tap-water  for  one  to  three  minutes,  dried  with 
filter-paper,  and  examined  for  the  Negri  bodies. 

Or  the  moist  films  may  be  fixed  in  methyl  alcohol,  and  without 
drying  stained  for  one  minute  in  a  mixture  of  10  c.c.  distilled  water, 
3  drops  of  a  saturated  alcoholic  solution  of  basic  fuchsin,  and  2  c.c. 
of  Loffler's  methylene-blue.  Eosin-methylene-blue  mixtures  may 
also  be  used. 

The  cytoplasm  of  the  bodies  stains  orange,  pink,  red,  or  magenta, 
the  central  nuclei  are  granular,  and  appear  bluish  or  purplish. 

Luzzani  considers  that  the  Negri  bodies  can  generally  be  well 
seen  in  teased  up  fresh  material  unstained.  It  is  stated  that  structures 
resembling  the  Negri  bodies  may  be  present  in  the  brain  after  death 
from  snake-bite.  s  < 

2.  If  the  Negri  bodies  cannot  be  detected,  inoculation  should 
be  performed.  The  brain  should  be  removed  as  soon  as  possible, 
and  if  it  cannot  be  manipulated  immediately,  should  be  placed 
in  sterile  glycerin.  From  the  middle  of  the  floor  of  the  fourth 
ventricle  a  small  piece  about  the  size  of  a  pea  is  removed  ;  this  is 
triturated  and  thoroughly  emulsified  in  a  sterile  watch-glass  by 
means  of  a  sterile  glass  rod  with  a  bulbous  end,  a  little  sterile  broth 
being  used  to  make  the  emulsion,  and  sufficient  being  added  to 
measure  about  10  c.c.  The  hair  on  the  head  of  a  good-sized  rabbit 
is  cut  close,  the  animal  is  anaesthetised  with  ether,  the  skin  on  the 
scalp  reflected  and  a  trephine  hole  made  through  the  skull.  The 
centre  of  the  trephine  hole  should  be  in  the  middle  line,  and  on 


INFANTILE  PARALYSIS  543 

the  line  drawn  between  the  posterior  corners  of  the  eyes,  the 
diameter  of  the  trephine  being  about  ^  inch.  A  little  of  the 
emulsion  is  drawn  up  in  a  small  syringe,  having  a  fine  needle,  and 
two  or  three  drops  are  injected  beneath  the  dura  mater.  The 
operation  is  carried  out  with  antiseptic  precautions,  the  wound 
closed,  and  a  little  wool  and  collodion  dressing  applied. 

If  the  material  injected  be  from  a  rabid  animal,  the  first  symptoms 
will  be  noticed  in  from  ten  to  fourteen  days.  The  inoculated  animal 
loses  control  over  its  hind  legs  and  throws  them  about  peculiarly 
when  running.  This  increases,  and  in  another  day  or  so  the 
animal  is  apt  to  fall  when  running,  and  in  another  day  or  two  the 
hinder  extremities  become  paralytic,  and  the  animal  is  unable  to 
move,  and  dies  shortly.  The  onset  of  symptoms  is  hardly  ever 
delayed  beyond  twenty-one  days. 

Van  Gehuchteri's  method. — The  ganglion  is  placed  in  absolute 
alcohol  for  twelve  hours,  the  alcohol  being  changed  once  ;  it  is  then 
embedded,  and  sections  are  cut.  These  are  stained  for  five  minutes 
in  Nissl's  methylene-blue  and  mounted.  Or  the  material  may  be 
fixed  in  10  per  cent,  formalin  before  staining.  The  capsular  changes 
are  best  shown  by  staining  with  haematoxylin  and  eosin. 

Babes'  method. — A  piece  of  the  medulla  or  cord  is  hardened  in 
alcohol  and  stained  with  anilin  red,  and  sections  are  prepared. 


Infantile  Paralysis  1 

Infantile  paralysis  or  acute  anterior  poliomyelitis  occurs 
sporadically  and  also  in  epidemics. 

Various  organisms  have  been  described  in  this  disease, 
but  recent  researches,  particularly  by  Levaditi,  Land- 
steiner,  and  Flexner,  have  proved  that  the  virus  is  a 
filter-passer. 

Injection  of  emulsions  of  the  affected  cord  into  the 
brain,  spinal  cord,  peritoneal  cavity,  and  blood-stream 
of  monkeys  reproduces  the  disease  with  the  same  clinical 
and  pathological  features  as  in  man.  The  disease  can 
be  carried  on  from  monkey  to  monkey  by  inoculation, 

1  See  Levaditi,  Journ.  Roy.  Inst.  of  Public  Health,  vol.  xix,  1911,  pp.  1 
and  65  (Bibliog.)  :  Flexner  and  others,  Journ.  Amer.  Med.  Assoc., 
1910-1911. 


544  A  MANUAL  OF  BACTERIOLOGY 

but  does  not  seem  to  be  transmissible  to  other  animals. 
The  salivary  and  some  of  the  lymphatic  glands  contain 
the  virus. 

Flexner  has  observed  a  case  of  spontaneous  infection 
in  the  monkey,  and  found  that  the  naso-pharyngeal 
mucosawas  infective,  so  that  this  is  probably  the  channel  of 
infection  in  man.  Flies  belonging  to  the  genus  Stomoxys 
are  stated  to  be  capable  of  transmitting  infection.  Human 
cerebro-spinal  fluid  was  not  found  infective  in  some 
instances,  but  monkey  cerebro-spinal  fluid  is  infective 
(infectivity  in  this  case  may  depend  on  the  stage  of  the 
disease). 

Human  ascitic  fluid  inoculated  with  the  filtered  fluid 
from  emulsions  of  cord  became  turbid,  but  no  organism 
could  be  detected  microscopically,  and  the  culture  can 
be  carried  on  from  tube  to  tube  (Flexner  and  Noguchi). 
Monkeys  which  have  recovered  from  an  attack  are  refrac- 
tory to  inoculation.  A  certain  degree  of  active  immunity 
may  be  established  by  subcutaneous  injection  of  the  virus. 
The  serum  of  immunised  and  recovered  animals  possesses 
considerable  neutralising  power  for  the  virus.  Attempts 
are  now  being  made  to  prepare  a  curative  serum. 

Some  cases  of  the  acute  ascending  paralysis  of  Landry 
may  be  forms  of  this  disease  (see  also  p.  535). 

Buzzard,  from  a  case  of  the  latter  disease,  isolated  a 
coccus  which  induced  a  rapidly  spreading  palsy  on  sub- 
dural  inoculation  into  rabbits. 

Typhus  Fever l 

Many  organisms  have  been  described  in  this  disease. 
Nicolle,  in  Tunis,  has  found  that  typhus  fever  of  man 
is  communicable  to  the  chimpanzee  by  inoculation  and 
from  the  anthropoid  to  the  Chinese  bonnet  monkey. 

1  See  Hewlett,  Practitioner,  July  1911,  p.  112  (Refs.). 


TYPHUS  FEVER  545 

Nicolle  and  Conseil  have  found  it  possible  directly  to  infect 
the  Macacus  sinicus  and  rhesus  monkeys  from  human 
cases. 

Nicolle  ascertained  that  the  blood  is  virulent  from  the 
commencement  of  infection  and  continues  so  until  the 
day  after  the  temperature  becomes  normal.  The  dog  and 
rat  are  quite  refractory.  The  disease  appears  to  be  trans- 
mitted by  the  body-louse  (P.  vestimenti),  not  by  the  flea, 
as  suggested  by  Matthew  Hay. 

The  blood  from  a  mild  case  does  not  produce  immunity 
on  injection,  nor  does  a  mild  attack  itself  induce  any 
appreciable  immunity.  On  the  other  hand  a  severe 
infection  induces  considerable  immunity.  Nicolle  and 
Jseggy  have  not  detected  any  microbe  in  affected  persons 
or  animals.  As  the  polymorphonuclear  leucocytes  suffer 
considerably  during  the  attack,  undergoing  fragmentation 
of  the  nucleus  and  necrosis,  it  is  suggested  that  the  micro- 
organism may  be  intra-leucocytic. 

Other  researches  have  been  carried  out  in  America  on 
the  typhus  of  Mexico,  known  locally  as  "  Tabardillo." 
Anderson  and  Goldberger  first  showed  that  the  Macacus 
rhesus  monkey  could  be  directly  infected  with  Mexican 
typhus.  Ricketts  and  Wilder  have  confirmed  this,  and 
find  that  typhus  blood  is  not  infective  if  passed  through  a 
Berkefeld  filter,  indicating  that  the  micro-organism  is  of 
appreciable  size.  They  also  find  that  the  disease  is  con- 
veyed by  the  body-louse,  and,  moreover,  that  the  infection 
is  hereditary  in  the  louse,  the  second  generation  of  lice 
derived  from  infected  lice  apparently  being  still  infective. 
Neither  bugs  nor  fleas  conveyed  the  disease. 

In  the  blood  of  typhus  patients  Ricketts  and  Wilder 
detected  a  small  bacillus,  measuring  2  /UL  in  length  by  O6  yu, 
in  breadth,  tending  to  stain  at  the  poles  and  belonging  to 
the  group  of  the  hsemorrhagic  septicsemic  bacteria.  It  is 
cot  numerous,  and  is  found  from  the  seventh  to  the  twelfth 

35 


546  A  MANUAL  OF  BACTERIOLOGY 

day  of  the  disease.  It  is  also  found  in  infected  lice,  but 
could  not  be  cultivated.  A  similar  micro-organism  was 
also  observed  in  Mexican  typhus  blood  by  Gavino  and 
Girard,  and  by  Campbell,  and  the  latter  also  finds  that 
the  blood  is  not  infective  if  passed  through  a  Chamberland 
F  filter. 

Ricketts  and  Wilder  also  discuss  the  relationship  between 
typhus  fever  and  Rocky  Mountain  spotted  fever.1  Some 
years  ago  Wilson  and  Chowning  made  observations  on  a 
typhus-like  fever  occurring  in  limited  tracts  of  country 
near  the  Rocky  Mountains  and  ascribed  it  to  a  Piroplasma. 
Subsequent  research,  however,  failed  to  confirm  this, 
though  the  disease  appears  to  be  conveyed  by  a  tick,  and 
not  by  fleas,  lice,  etc.  There  are  clinical  differences 
between  typhus  and  Rocky  Mountain  spotted  fever  ;  more- 
over, the  guinea-pig  is  susceptible  to  the  spotted  fever 
but  not  to  typhus,  and  a  monkey  immunised  to  typhus  is 
susceptible  to  spotted  fever.  Ricketts  believes  that  the 
spotted  fever  is  due  to  a  bacillus  which  can  be  found 
in  the  ovary  of  the  tick  and  is  agglutinated  by  the  serum 
in  dilutions  of  1-500. 

Cathoire  has  made  observations  on  complement  fixation 
in  typhus.  Using  as  an  antigen  an  alcoholic  extract  of 
typhus  spleen,  marked  complement  fixation  was  obtained 
with  the  serum  of  typhus  cases. 


Yellow  Fever 

As  far  back  as  1889  Sternberg  described  a  bacillus — 
"  Bacillus  X  " — in  yellow  fever,  a  facultative  anaerobic 
organism,  very  pathogenic  to  rabbits.  In  1897  Sanarelli  2 
described  his  Bacillus  ictero'ides,  which  later  investigation 

1  The  name  is  an  unfortunate  one,  for  this  disease  is  quite  distinct 
from  "  spotted  fever  " — epidemic  cere bro -spinal  meningitis. 

2  Ann.  de  VInst.  Pasteur,  xi,  1897,  pp.  443,  673,  and  753. 


YELLOW  FEVER  547 

has  proved  to  be  an  organism  belonging  to  the  Gartner 
group  (see  p.  371). 

Reed  and  Carroll 1  critically  examined  the  B.  ictero'ides 
and  its  relation  to  yellow  fever.  Their  conclusions  were 
that  the  Bacillus  X  belongs  to  the  colon  group,  the  B. 
ictero'ides  to  the  Gartner  group,  that  the  B.  ictero'ides  and 
hog- cholera  bacillus  produce  the  same  lesions  in  animals 
and  mutually  protect  against  each  other,  that  the  B. 
ictero'ides  causes  in  swine  all  the  symptoms  and  lesions  of 
hog  cholera,  and  that  the  blood  of  hog  cholera  agglutinates 
the  B.  ictero'ides  in  a  much  more  marked  degree  than  does 
the  blood  of  yellow  fever. 

Reed,  Carroll,  and  Agramonte,2  having  thus  shown  the 
etiological  position  of  the  B.  ictero'ides  to  be  untenable, 
directed  their  attention  to  the  transference  of  yellow  fever 
through  the  agency  of  mosquitoes.  Finlay,  of  Havanah, 
suggested  many  years  ago  that  yellow  fever  might  be 
propagated  through  the  intermediary  of  a  mosquito — 
Stegomyia  calopus  (fasciata) — and  with  this  species  these 
investigators  worked.  They  allowed  mosquitoes  to  bite 
yellow-fever  patients  at  various  stages  of  the  disease,  and 
the  infected  mosquitoes  were  subsequently  allowed  to  bite 
eleven  individuals,  two  of  whom  contracted  yellow  fever. 
It  is  true  this  is  not  a  very  convincing  experiment,  but 
it  is  to  be  noted  that  during  the  period  of  fifty-seven  days 
among  a  population  of  1400  non- immune  Americans  there 
were  only  three  cases  of  yellow  fever,  and  that  two  of  these 
had  been  bitten  by  contaminated  mosquitoes  within  five 
days  of  the  commencement  of  their  attacks.  The  matter 
was  put  to  the  further  test  of  experiment  in  the  following 
manner.3  Under  the  same  observers  a  camp  was  estab- 
lished with  several  tents  each  occupied  by  one  to  three 

1  Journ.  Exper.  Med.,  vol.  v,  pt.  iii,  p.  215. 

2  Philad.  Med.  Journ.,  October  27,  1900,  p.  790. 

3  Journ.  Amer.  Med.  Assoc.,  February  16,  1901,  p.  431. 


548  A  MANUAL  OF  BACTERIOLOGY 

non-immune  individuals,  and  precautions  were  taken  to 
prevent  the  introduction  of  yellow  fever  from  outside. 
Five  individuals  were  bitten  by  infected  mosquitoes,  and 
four  out  of  the  five  contracted  yellow  fever,  no  other 
occupants  of  the  camp  being  attacked  by  the  disease. 
Subsequently  several  non-immune  individuals  were  exposed 
to  yellow  fever  infection  from  soiled  linen,  yellow-fever 
discharges,  etc.,  in  a  mosquito-proof  hut  from  which 
mosquitoes  were  excluded,  with  entirely  negative  results. 
These  experiments  prove,  therefore,  that  yellow  fever  is 
conveyed  by  mosquitoes  only,  and  further  work  by 
Americans  and  Cubans,  and  by  French  and  Brazilian 
Commissions,  has  entirely  confirmed  these  researches 
and  conclusions.  It  has  been  found  that  to  convey  infec- 
tion, it  is  necessary  for  the  mosquitoes  to  bite  the  patient 
during  the  first  three  or  four  days  of  the  illness,  but  they 
do  not  become  infective  until  about  the  twelfth  day  after 
feeding,  and  then  retain  their  infectivity  indefinitely. 
All  these  facts  point  to  a  protozoon  as  being  the  causative 
organism,  but  none  has  been  found  with  certainty. 

The  Americans  have  shown  that  the  blood-serum  after 
filtration  through  a  porcelain  filter  is  still  infective ;  the 
organism,  therefore,  is  probably  ultra-microscopic,  at  least 
at  one  stage.  Seidelin  1  describes  extremely  small  rounded 
bodies  with  a  minute  chromatin  point  and  feebly  staining 
protoplasm,  without  pigment,  in  the  blood  corpuscles. 
Somewhat  similar,  but  larger,  bodies  may  also  be  present 
in  the  organs  and  free  in  the  plasma.  Macfie  and  Johnston  2 
state  that  they  have  found  elements  similar  to  those 
described  by  Seidelin  in  the  red  corpuscles  in  practically 
every  case  of  yellow  fever  examined. 

1  Journ.  Pathol.  and  Bacterial,  vol.  xv,  1911,  p.  282. 

2  Proc.  Roy.  Soc.  Med.,  vii,  No.  3,  1914  (Med.  Sec.),  p.  49. 


DENGUE  AND  PHLEBOTOMUS  FEVER     549 


Dengue 

No  organism,  bacterium  or  protozoon,  has  been  demon- 
strated in  this  disease.  The  intra-venous  inoculation  of 
filtered  dengue  blood  into  healthy  individuals  is  followed 
by  an  attack  ;  the  organism  is  therefore  probably  ultra- 
microscopic.  The  disease  can  be  transmitted  by  a  mos- 
quito, Culex  fatigens,  and  this  is  probably  the  common 
mode  of  infection.1 


Phlebotomus  Fever 

A  fever  of  short  duration  (three  days)  occurs  in  South 
Austria,  the  malady  being  somewhat  like  dengue.  It  is 
known  locally  as  "  pappataci,"  and  an  apparently  identical 
disease  has  been  described  by  Birt 2  in  Malta  under  the 
name  of  "  phlebotomus  fever."  Investigation  has  shown 
that  this  disease  is  conveyed  by  the  bite  of  a  dipterous  fly, 
the  sand-fly  (Phlebotomus  pappatasii).  "  Canary  fever," 
"  Shanghai  fever,"  "  Chitral  fever,"  and  the  seven  days 
continued  and  "  sand-fly  "  fevers  of  India  are  probably 
of  the  same  nature.  The  virus  in  phlebotomus  fever  passes 
through  a  Berkefeld  filter. 

Further  research  must  decide  whether  these  and  dengue 
are  distinct  diseases  or  whether  they  are  all  manifestations 
of  dengue. 

Variola  and  Vaccinia 

The  specific  contagia  of  these  two  diseases  appear 
to  be  filter-passers. 

Variola  is  inoculable  on  man  the  calf  and  the  monkey, 
vaccinia  on  the  rabbit  in  addition. 

1  Ashburn  and  Craig,  Philippine  Journ.  of  Science,  vol.  ii,  1907,  p.  93. 

2  Journ.  Roy.  Army  Hed.  Corps,  August  1910. 


550  A  MANUAL  OF  BACTEKIOLOGY 

A  large  number  of  observations  have  been  made  with 
vaccine  lymph,  but  no  distinctive  bacterium  has  been 
obtained  except  by  Klein  and  Copeman.  Usually  the 
ordinary  pyogenic  organisms  and  many  saprophytic  forms 
can  alone  be  isolated.  Klein  observed  the  presence  of  a 
bacillus  in  vaccinia,  which  was  subsequently  more  fully 
studied  by  Copeman.1  It  was  found  in  vaccine  vesicles 
at  an  early  stage,  but  at  maturation  could  no  longer  be 
detected.  It  is  a  very  fine  bacillus,  and  these  observers 
were  unable  to  cultivate  it.  Subsequently  Copeman 
found  a  similar  organism  in  variola,  and  succeeding  in 
cultivating  the  bacillus  from  both  sources  in  eggs,  and  from 
such  egg-cultures  was  able  to  inoculate  calves.  Klein,2  by 
storing  variola  crusts  in  50  per  cent,  glycerin  and  so  getting 
rid  of  the  saprophytic  forms,  has  cultivated  an  organism 
which  he  terms  the  Bacillus  albus  variolce.  Morphologically 
it  closely  resembles  the  bacillus  observed  in  vaccine  lymph  ; 
it  forms  small  white,  opaque,  coherent  colonies  on  agar, 
but  grows  very  feebly  on  gelatin.  Involution  forms  occur, 
and  it  seems  to  belong  to  the  group  of  diphtheria  and  xerosis 
bacilli.  On  inoculation  into  calves  some  approach  to, 
but  not  typical,  vaccinia  was  produced.  Moreover,  the 
inoculated  calves  were  not  immune  to  subsequent  vaccina- 
tion. Copeman  3  inoculated  glycerinated  vaccine  lymph 
in  which  the  extraneous  organisms  had  died  out  into 
collodion  capsules  filled  with  beef  broth  and  inserted  them 
in  the  peritoneal  cavity  of  rabbits,  and  observed  zooglcea 
masses  made  up  of  bodies  resembling  spores  which  he 
regards  as  the  resting  stage  of  the  specific  microbe. 

De  Korte  finds  that  the  vesicles,  both  in  variola  and  in 
vaccinia,  are  sterile  before  maturation,  and  regards  the  bac- 
terial forms  that  have  been  isolated  as  secondary  infections. 

1  Milroy  Lectures  on  Vaccination,  1898. 

2  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1896-97,  p.  267. 

3  Brit.  Med.  Journ.,  1901,  vol.  i,  p.  450. 


VARIOLA  AND  VARICELLA  551 

The  failure  to  isolate  a  bacterial  form  has  induced  many 
observers  to  seek  for  a  parasitic  protozoon  in  variola  and 
vaccinia.  L.  Pfeiffer  in  1887  observed  roundish  or  ovoid 
bodies  in  the  lymph  in  both  diseases,  which  he  regarded 
as  sporozoa.  Guarnieri  found  small  bodies,  about  half 
the  size  of  the  nucleus,  in  the  epithelial  cells  of  the  skin 
in  the  prepustular  stage  of  variola  (Cytoryctes  variola). 
Small  shining  amoeboid  bodies  were  also  noticed  in  the 
epithelial  cells  of  the  corneae  of  guinea-pigs  inoculated 
with  vaccine  lymph.  L.  Pfeiffer  confirmed  Guarnieri's 
work,  and  also  described  these  amcebiform  parasites  in 
the  blood  in  variola  and  vaccinia,  and  of  vaccinated  calves. 
J.  Clarke,  and  RutTer  and  Plimmer  in  this  country  described 
somewhat  similar  appearances.  Ruffer  and  Plimmer 
describe  the  supposed  protozoon  as  a  small  round  body, 
about  3  JUL  in  diameter,  lying  within  a  clear  vacuole  in 
the  protoplasm  of  the  epithelial  cell. 

Councilman,  Magarth,  Brinkerkoff,  Tyzzer,  and  Calkins  l 
in  America  have  found  the  Guarnieri  body  in  variola  and 
vaccinia  in  man  and  animals,  and  regard  it  as  a  protozoon 
and  the  causal  agent  of  these  diseases. 

Ogata  found  bodies  which  he  regards  as  parasitic  pro- 
tozoa and  the  causative  agent  of  the  disease  in  variolous 
and  vaccine  lymph.  Reed  likewise  observed  small  granular 
amoeboid  bodies  having  a  diameter  of  about  one-third 
that  of  a  red  blood- corpuscle,  similar  apparently  to  those 
described  by  L.  Pfeiffer,  in  the  blood  of  vaccinated  children 
and  monkeys,  but  also  observed  them — and  this  is  impor- 
tant— occasionally  in  the  blood  of  normal  children  and 
monkeys. 

Funck,  Roger  and  Weil,  and  Calmette 2  have  also 
observed  various  bodies  and  retractile  granules  in  lymph. 

1  Journ.  Med.  Research,  vol.  xi,  1904,  p.  173  -^Philippine  Journ.  of 
Science,  vol.  i,  1906,  p.  239.  $&       ;  1*\ 

2  Ann.  de  Vlnst.  Pasteur,  xv,jl901.  No.  3,  p.  161, 


552  A  MANUAL  OF  BACTERIOLOGY 

The  monkey  and  rabbit  are  both  susceptible  to  vaccinia ; 
in  the  latter  animal  the  pustules  are  mature  on  the  third 
day  and  immunity  is  acquired  by  the  sixth  day. 

Ferroni  and  Massari  state  that  appearances  similar  to 
those  described  by  Guarnieri  can  be  obtained  in  cornese 
inflamed  by  croton  oil  or  Indian  ink,  and  therefore  believe 
that  the  so-called  parasites  are  derived  from  the  nuclei 
or  from  emigrated  leucocytes.  Salmon  considers  that  the 
so-called  parasites  in  vaccinia  and  variola  are  more  or  less 
condensed  balls  of  chromatin  of  extra- epithelial  origin 
derived  from  the  migratory  polynuclear  leucocytes. 
According  to  von  Prowazek  these  cell  inclusions  (the 
Guarnieri  bodies,  etc.)  in  this  and  other  conditions  (e.q. 

'I  \      ij 

scarlatina)  are  not  parasites,  but  consist  of  plastin  and 
nuclease,  and  are  derived  from  the  cells  in  which  they  occur. 

De  Korte  1  has  observed  in  the  variolous  and  vaccine 
vesicles  before  maturation  large  amoeboid  bodies  (10  /u), 
which  he  believes  to  be  protozoa  (Sporidium  vaccinate). 
In  vaccine  lymph  refractile  motile  granules  occur  in 
abundance,  believed  by  De  Korte  to  be  spores. 

Fornet  2  by  treating  variola  or  vaccine  lymph  with  ether 
finds  a  stage  when  all  the  bacteria  are  killed  but  the  specific 
virus  is  uninjured.  By  inoculating  this  etherised  lymph 
into  nutrient  broth  and  keeping  at  37°  C.,  the  broth  culture 
inoculated  in  man  produces  typical  vesicles  even  after 
two  months  incubation,  and  moreover  the  culture  can 
be  carried  on  from  tube  to  tube.  In  the  broth,  minute 
rounded  bodies  can  be  detected  which  may  be  the  specific 
micro-organism. 

The  relationship  of  vaccinia  to  variola  has  been  a  very 
vexed  question.  With  few  exceptions  (Ceely,  Hime, 
Simpson,  Klein,  King,  Copeman)  attempts  to  inoculate 

1  Trans.  Path.  Soc.  Lond.,  vol.  Ivi,  1905,  p.  172. 

2  Trans.  XVIIih  Internal.  Cong.  Med.  Lond.,  1913,  Sect,  iv,  pt.  ii, 
p.  119. 


VARIOLA  AND  VARICELLA  553 

variola  on  the  calf  have  failed.  In  the  successful  cases 
the  lymph  obtained  from  the  calf  has,  on  inoculation  upon 
children,  produced  typical  vaccinia  without  any  untoward 
results.  The  positive  results  obtained  by  the  inoculation 
of  variolous  material  being  so  few,  a  doubt  arises  whether 
in  these  cases  there  may  not  have  been  some  fallacy,  such 
as  accidental  contamination  with  vaccinia.  Simpson, 
however,  performed  his  experiments  within  the  precincts 
of  a  smallpox  hospital  and  away  from  possible  vaccine 
infection,  and  Copeman  *  found  that  variola  may  be  readily 
inoculated  upon  monkeys,  and  after  several  passages 
through  these  animals  is  easily  inoculable  upon  the  calf. 
He  suggests,  therefore,  that  vaccinia  in  the  calf  was  origin- 
ally due  to  infection  with  inoculated  smallpox,  so  prevalent 
at  the  time  of  Jenner's  discovery.  A  somewhat  parallel 
instance  of  the  attenuation  of  a  virus  by  passage  through 
another  animal  is  recorded  by  Sticker  and  Marx  in  the 
case  of  birdpox,  which  produces  an  extensive  smallpox- 
like  eruption  in  fowls  and  pigeons.  In  fowls  and  in  pigeons 
the  virus  retains  its  pathogenic  properties  for  each  bird 
unaltered  for  any  number  of  inoculations,  but  the  pigeon 
strain,  after  a  few  inoculations  into  fowls,  completely  loses 
its  virulence  for  the  pigeon.  There  seems  little  doubt, 
therefore,  that  vaccinia  is  modified  variola,  and  the  rationale 
of  vaccination  rests  upon  a  scientific  basis. 

The  preparation  of  vaccine  lymph  is  fully  described  by  Blaxall.2 
Calves  are  vaccinated  with  lymph  under  aseptic  precautions,  and 
five  days  later  the  contents  of  the  vesicles  are  scraped  off,  the  pulp 
is  triturated  in  a  machine,  and  is  then  placed  in  six  times  its  weight 
of  sterilised  50  per  cent,  pure  glycerin  in  distilled  water,  and  stored 
for  about  a  month  in  test-tubes,  until  agar  cultivations  show  that 
extraneous  bacteria  have  died  out,  when  it  is  issued  for  use.  It 
remains  very  active  for  fifty  to  sixty  days,  after  which  it  begins  to 
deteriorate. 

1  Brit.  Med.  Journ.,  1901,  vol.  i,  p.  1134,  and  1901,  vol.  ii,  p.  1736. 

2  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1898-99,  p.  35. 


554  A  MANUAL  OF  BACTERIOLOGY 

Green  *  rapidly  prepares  vaccine  lymph  by  killing  off  the  extra- 
neous organisms  with  chloroform  vapour. 

Blaxall  2  has  more  recently  used  oil  of  cloves  as  a  sterilising  agent 
in  the  preparation  of  calf  lymph. 


Malignant  Disease 

The  analogies  between  carcinoma  and  sarcoma  and  many  infec- 
tive diseases  have  led  investigators  to  search  for  micro-organisms 
in  these  conditions. 

Bacteria  have  been  repeatedly  looked  for,  but  Shattock  was 
unable  to  isolate  any  bacterial  form  from  malignant  disease.  Doyen 
isolated  a  micrococcus  (M.  neoformans,  p.  232),  but,  though  fre- 
quently present,  it  is  not  causative. 

A  great  impetus  was  given  to  the  study  of  parasites  in  malignant 
disease  by  the  publication  of  a  paper  by  Russell.  He  observed, 
by  certain  methods  of  staining,  small  corpuscles  within  the  epithelial 
cells.  They  were  spherical  in  shape,  4  to  10  /z  in  diameter,  occurring 
singly  or  in  groups,  were  apparently  homogeneous,  and  surrounded 
by  a  capsule.  Russell  regarded  these  structures  as  belonging  to 
the  "  sprouting  fungi  "  (Blastomycetes),  and  they  have  since  been 
known  by  the  name  of  "  fuchsin  bodies  "  or  "  Russell's  corpuscles." 

Subsequently  structures  were  observed  within  the  epithelial 
cells  of  carcinoma  which  were  regarded  by  many  investigators  as 
parasitic  protozoa.3  These  structures  are  round  or  ovoid,  2  ^  to 
10  fj,  in  diameter,  with  a  very  distinct  outline,  as  though  encapsuled, 
and  clear  refractile  contents  in  which  is  a  smaller  body  of  variable 
size  analogous  to  a  nucleus  (Fig.  66,  a).  Occasionally  the  refractile 
contents  present  a  radial  striation  or  a  granulation. 

These  bodies  are  usually  single,  but  may  number  as  many  as 
eight  or  ten,  and  sometimes  they  invade  the  epithelial  nucleus. 
The  Ruffer's  or  Plimmer's  body,  however,  is  a  structure  probably 
analogous  to  the  archoplastic  vesicle  of  the  cells  of  reproductive 
tissue  (Fig.  66,  6).  Save  for  the  presence  of  these  structures, 
there  is  no  proof  that  protozoa  are  present  in,  or  are  the  cause  of, 
carcinoma. 

Another  hypothesis  of  the  nature  of  malignant  disease  is  that 
it  is  due  to  a  blastomycetic  infection  (see  p.  462). 

Washbourn  and  others  have  observed  infective  venereal  tumours 

1  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1900-01,  p.  639. 

2  Ibid.  191 1-12,  p.  361. 

3  See  Ruffer  and  Walker,  Journ.  Path,  and  Bact.,  vol.  i,  1893,  p.  395. 


MALIGNANT  DISEASE  555 

in  dogs.     These  have  been  stated  to  be  sarcomata,  but  are  probably 
granulomata. 

Malignant  disease  occurs  in  all  classes  of  vertebrates,  and  is 
generally  inoculable  on  an  animal  of  the  same  species  as  that  from 
which  it  is  derived,  but  not  on  other  animals.  The  carcinoma  of 
mice  has  been  the  subject  of  much  investigation  of  late.  In  the 
writer's  opinion,  the  trend  of  recent  research  is  to  show  that  malig- 
nant disease  is  not  due  to  a  micro-parasite,  but  is  derived  from  the 


FIG.  66. — a,  Buffer's  or  Plimmer's  body  in  a  cancer-cell ; 
b,  the  archoplastic  vesicle  in  spermatid  of  mouse.  (After 
Farmer,  Moore,  and  Walker.) 

irresponsible  division  of  cells  of  the  normal  or  of  embryonic  tissues.1 
If  there  be  a  parasite,  in  all  probability  it  is  intra-cellular,  like  the 
organism  of  plant  cancer  (Bacterium  tumefaciens)  described  by 
Erwin  Smith.2 

The  molluscum  bodies  have  likewise  been  regarded  as  parasitic 
(coccidial)  in  nature,  but  with  them  also  inoculation  and  cultivation 
experiments  have  failed.  The  virus  is  stated  to  be  a  filter-passer, 
as  is  also  the  case  with  bird  molluscum. 

Certain  malignant-like  tumours  of  birds  are  also  filter -passers, 
e.g.  chicken  sarcoma. 

1  For  further  information  consult  Pathology,   General  and  Special, 
ed.  3,  R.  T.  Hewlett  (Churchill,  1912). 

2  Trans.    XVIIth  Internal.  Cong.  Med.  Land.,  1913,  Sect,  iii,  pt.  ii, 
p.  281. 


CHAPTER  XX 

SOME  DISEASES  NOT  PREVIOUSLY  REFERRED  TO,  WITH 
A  DISCUSSION  OF  THEIR  CAUSATION  —  MICRO- 
ORGANISMS OF  SKIN  AND  MUCOUS -MEMBRANES 


APPENDICITIS. — The  following  Table  *  shows  the  usual  kinds  and 
relative  frequency  of  the  infections  in  appendicitis  : 


Micro-organism. 

Acute 
appendicitis. 

Chronic 
appendicitis. 

Bacillus  coli  in  pure  culture 

70  per  cent. 

90  per  cent. 

,,            with  staphylococci 

15 

6 

„                 „    streptococci   . 

7 

Very  rare. 

Staphylococci  alone 

4 

1  per  cent. 

Streptococci          ,, 

Very  rare. 

Very  rare. 

Other  organisms  or  combinations 

4  per  cent. 

3  per  cent. 

It  is  not  improbable  that  in  a  still  greater  percentage  of  cases 
a  mixture  of  organisms  is  present  at  first,  the  Bacillus  coli  subse- 
quently crowding  out  the  other  forms.  The  Bacillus  proteus, 
B.  pyocyaneus,  and  B.  Welchii  also  occasionally  occur. 

Castellani 2  describes  a  bacillus,  pathogenic  to  guinea-pigs, 
isolated  from  a  case  of  gangrenous  appendicitis.  Morphologically 
it  resembled  the  Shiga-Kruse  dysentery  bacillus,  and  was  non- 
motile,  produced  acid  and  gas  in  glucose  and  maltose  and  curdled 
milk,  but  did  not  ferment  mannite,  lactose,  and  sucrose. 

BEBI-BEBI. — Various  observers  have  attempted  to  cultivate  a 
micro-organism  in  this  disease.  Cocci  have  been  described  by 
Pekelharing  and  Winkler,  Hunter,  Okata  and  Kokubo,  a  sporing 
bacillus  by  Rost,  and  Hamilton  Wright  suggests  that  the  disease 
is  due  to  an  intoxication,  the  result  of  a  gastro- duodenal  infection 

1  Battle  and  Corner,  Diseases  of  the  Vermiform  Appendix,  1904. 

2  Brit.  Med.  Journ.,  1907,  vol.  i,  p.  1513. 

556 


CONJUNCTIVITIS  557 

with  a  large  Gram-positive  bacillus  (unisolated).  Daniels  suggested 
that  the  epidemiology  of  the  disease  is  best  explained  on  the  hypo- 
thesis of  a  protozoan  infection  conveyed  by  lice.  The  writer  and 
De  Korte  *  also  suggest  a  protozoan  infection,  the  organism  perhaps 
being  eliminated  in  the  urine. 

Other  views  are  that  beri-beri  may  be  a  peripheral  neuritis  due 
to  arsenical  poisoning,  or  that  it  is  caused  by  the  absence  of  certain 
nutritive  elements  from  polished  rice.  The  evidence  in  favour  of 
the  latter  view  seems  to  be  accumulating,  and  it  has  been  found 
that  essential  nutritive  constituents  (vitamines  ?)  are  present  in  the 
husk  of  rice  which  is  removed  in  polishing. 

BRONCHITIS. — Ritchie 2  concludes  that  acute  bronchitis  is  an 
infective  disease,  but  is  not  due  to  any  one  specific  organism,  the 
most  important  causal  bacteria  being  the  S.  pneumonice  and  strep- 
tococci. In  every  case  of  acute  bronchitis  numerous  pathogenic 
bacteria  are  present  in  the  bronchi,  which  are  usually  sterile  in 
health.  The  commonest  organisms  are  B.  pneumonice,  B.  influenzce, 
and  M.  catarrhalis.  Spirochaetes  are  present  in  some  forms  of 
tropical  bronchitis  ;  in  others  Castellani  has  described  oidium- 
like  and  yeast-like  organisms. 

CHANCRE,  SOFT. — An  extremely  small  bacillus,  first  described 
by  Ducrey,3  has  been  found  in  the  ulcers  and  buboes.  It  has  not 
been  inoculated  successfully  on  animals,  but  can  be  inoculated 
from  a  chancre,  experimentally,  from  man  to  man.  The  bacillus 
does  not  stain  by  Gram's  method,  and  can  be  cultivated  on  blood 
agar,  on  which  it  forms  shining  greyish  colonies  1  mm.  in  diameter, 
or  in  guinea-pig  blood.4 

CONJUNCTIVITIS. — Conjunctivitis  is  of  several  varieties: 

(a)  Acute  contagious  conjunctivitis,  due  to  the  Koch-Weeks 
bacillus.  This  is  a  slender,  non-motile  organism,  1-1-5  ^  in  length, 
occurring  singly  or  in  pairs,  both  free  and  within  the  pus-cells. 
It  is  decolorised  by  Gram's  method,  and  is  difficult  to  cultivate, 
growing  best  on  a  serum-agar  mixture,  on  which  it  forms  small, 
punctiform  transparent  colonies.  It  is  hardly  pathogenic  to 
animals,  but  in  man  sets  up  a  typical  acute  conjunctivitis. 

(6)  Chronic  catarrhal  conjunctivitis,  due  to  the  Morax-Axenfeld 
diplo-bacillus.  This  organism  is  2  /j,  long  by  1  /M  broad,  is  not 
stained  by  Gram's  method,  and  can  be  cultivated  on  blood-serum 
which  is  liquefied,  or  serum  agar. 

1  Journ.  Trop.  Med.,  October  1,  1907,  p.  315. 

2  Journ.  Path,  and  Bact.,  vol.  vii,  No.  1,  p.  1. 

3  Comp.  Rend.  Congres  Internal,  de  Dermatologie  (Paris,  1889),  p.  229. 

4  Himmel,  Ann.  de  Vlnst.  Pasteur,  xv,  1901,  p.  928. 


558  A  MANUAL  OF  BACTERIOLOGY 

(c)  Gonorrhceal  conjunctivitis. 

(d)  Diphtheritic  conjunctivitis. 

(e)  Conjunctivitis  of  streptococcic  origin. 

(/)  Conjunctivitis  of  pneumococcic  origin. — Usually  in  children, 
and  accompanied  with  coryza  and  scanty  muco-purulent  discharge. 

(g)  Micrococci  (aureus  and  albus)  and  B.  coli  may  also  occasionally 
cause  conjunctivitis. 

DIARRHOEA  (SUMMER)  OF  INFANTS. — Booker,1  in  an  elaborate 
paper,  came  to  the  following  conclusions :  "  No  single  micro- 
organism is  found  to  be  the  specific  exciter  of  the  summer  diarrhoea 
of  infants,  but  the  affection  is  generally  to  be  attributed  to  the 
activity  of  a  number  of  varieties  of  bacteria,  some  of  which  belong 
to  well-known  species,  and  are  of  ordinary  occurrence  and  wide 
distribution,  the  most  important  being  a  streptococcus  and  the 
Proteus  vulgaris." 

Lesage  obtained  a  bacillus  from  the  "  green  diarrhoea  "  of  infants 
which  he  believed  to  be  the  cause  of  this  complaint.  It  is  a  small, 
motile,  non -liquefy ing  bacillus,  producing  on  gelatin  a  whitish 
expanded  growth  with  crenated  margins,  and  giving  rise  to  a  green 
fluorescence  in  the  medium.  The  B.  pyocyaneus  may  be  an 
occasional  cause. 

In  cases  with  blood  and  mucus  in  the  stools,  the  B.  dysenteric^ 
(Shiga-Kruse  type)  has  been  found  to  be  present  in  America  and 
in  this  country.  In  London,  Morgan  has  isolated  in  a  number  of 
cases  a  bacillus  which  in  its  fermentation  reactions  is  nearly  allied 
to  the  hog-cholera  bacillus  (see  p.  372).  Lewis  2  found  that  non- 
liquefying  and  non-lactose-fermenting  bacilli  are  more  frequent  in 
the  faeces  of  children  suffering  from  diarrhoea  than  in  normal  children, 
and  believes  that  Morgan's  bacillus  has  a  causal  relationship  in  many 
cases.  Alexander  3  also  found  Morgan's  bacillus  more  frequent  in 
diarrhoea  cases  than  in  normal  children. 

Ralph  Vincent  ascribes  the  disease  (which  he  terms  "  zymotic 
enteritis  ")  to  the  ordinary  organisms  of  putrefaction  gaining  access 
to  milk  and  multiplying  and  causing  alterations  therein. 

The  stinking  motions  of  the  diarrhoea  of  children  have  been 
ascribed  to  the  action  of  organisms  belonging  to  the  Proteus  group, 
particularly  B.  proteus  (P.  vulgaris,  see  p.  621),  which  occurs  in 
putrefying  matter,  sewage,  and  in  the  intestine.  (This  organism 
may  also  cause  abscesses  and  cystitis,  and  a  form  of  meat  poisoning 

1  Johns  Hopkins  Hosp.  Reps.,  vol.  vi,  1897,  p.  159  (Bibliog.). 

2  Rep.  Med.  Loc.  Gov.  Board  for  1911-12,  p.  265,  and  ibid,  for  1912-13, 
p,  375. 

3  Ibid.  1911-12,  p.  288. 


DYSENTERY  559 

has  been  ascribed  to  its  action.)  Filtrates  of  cultures  were  found 
by  S.  Martin  to  produce  a  fall  of  temperature,  collapse,  and  diarrhoea 
in  rabbits. 

CANINE  DISTEMPER. — According  to  Galli- Valeric,1  this  is  caused 
by  a  bacillus  (B.  caniculce)  intermediate  in  character  between  the 
coli-typhoid  and  hsemorrhagic  septicaemic  groups  of  organisms. 
Torrey  and  Rahe  2  confirm  Ferry  and  M'Gowan's  observations  on 
a  bacillus  (B.  bronchisepticus)  present  in  distemper.  It  does  not 
ferment  any  sugars  and  litmus  milk  becomes  markedly  alkaline. 

Evidence  has  also  been  brought  forward  that  distemper  is  due 
to  a  filter  passer  (Carre).  Probably  the  term  "  distemper  "  may 
include  several  different  diseases. 

DYSENTERY. — Dysentery  must  be  regarded  as  a  term  applied 
to  a  series  of  clinical  symptoms  associated  with  colitis  which  is 
due  to  different  specific  agents.  There  are  at  least  two  forms  of 
the  disease,  one,  the  so-called  tropical  or  endemic  dysentery,  met 
with  especially  in  the  East,  and  characterised  by  chronicity,  a  ten- 
dency to  relapses,  amenability  to  treatment  with  ipecacuanha,  and 
the  occurrence  of  the  single  liver  abscess  as  a  sequela  ;  the  other, 
epidemic  dysentery,  met  with  in  all  parts  of  the  world,  particularly 
in  times  of  war  and  famine,  not  amenable  to  ipecacuanha,  and  not 
followed  by  liver  abscess.  There  are  also  probably  other  forms 
occurring  in  small  outbreaks  or  sporadically.  Tropical  dysentery 
is  due  to  the  Amoeba  coli,  which  is  found  abundantly  in  the  stools, 
especially  in  the  acute  stage,  and  also  in  the  liver  abscesses  (see 
p.  484). 

In  the  epidemic  dysentery  of  Japan  and  other  parts  of  the  world 
a  bacillus,  or  group  of  bacilli,  has  been  isolated  by  Shiga,  Flexner, 
Strong,  Kruse,  and  others.  This  is  the  B.  dysenteries  described 
at  p.  376. 

Coli-form  bacilli  have  been  isolated  from  cases  of  dysentery. 
Calmette  in  Tonkin  isolated  the  B.  pyocyaneus,  and  this  organism 
seems  to  have  been  the  cause  of  a  small  outbreak  in  New  York 
State  investigated  by  Lartigau.3  In  Japan,  Ogata  isolated  a  fine 
Gram-staining,  liquefying  bacillus  which  does  not  seem  to  have 
been  met  with  by  subsequent  observers.  Spirochaetes  have  been 
found  in  large  numbers  in  a  form  of  dysentery  occurring  in  Bordeaux. 

Vedder  and  Duval,4  as  a  result  of  the  study  of  a  number  of  cases 

1  Centr.  f.  Bakt.  (Ref.),  xli,  1908,  p.  563.     See  also  M'Gowan,  Journ. 
Palhol.  and  Bacterial.,  vol.  xv,  1911,  p.  372  (Bibliog.)  and  xvi,  p.  257. 

2  Journ.  Med.  Research,  xxvii,  1912,  p.  291  (Bibliog.). 

3  Journ.  Exper.  Med.,  vol.  iii,  No.  6,  p.  595. 

4  Ibid.  vol.  vi,  1902,  No.  2,  p.  181. 


560  A  MANUAL  OF  BACTERIOLOGY 

of  acute  dysentery  in  the  United  States,  conclude  that  the  disease, 
whether  sporadic,  "  institutional,"  or  epidemic,  is  due  to  the  B. 
dy sentence  of  Shiga. 

The  B.  dysenterice  (Shiga  type)  has  been  isolated  by  Eyre, 
McWeeney,  and  others  from  cases  of  ulcerative  colitis  or  asylums 
dysentery  in  the  British  Isles  (see  pp.  376-379). 

The  Balantidium  coli  (p.  507)  and  certain  parasitic  worms  may 
also  induce  a  dysenteric  condition. 

SKIN  DISEASES  :  Acne. — In  the  acne  pustules,  the  M.  pyogenes 
var.  aureus,  with  or  without  var.  albus,  is  almost  invariably  present, 
and  a  staphylococcic  vaccine  generally  acts  extremely  well.  In 
the  comedoes  a  Gram-positive,  Hofmann-like  bacillus  (B.  acnes) 
is  present  in  considerable  numbers,  and  may  be  the  cause  of  the 
comedo.  This  organism  was  cultivated  by  Fleming  on  a  neutral 
agar  to  which  glycerin  and  oleic  acid  are  added.  Siidmersen  and 
Thompson  *  cultivate  it  on  an  acid  ( +  40)  serum-agar.  The  organism 
is  anaerobic,  at  least  at  first,  and  will  grow  in  glucose-agar  stabs. 
In  culture  the  organism  is  diphtheroid.  A  vaccine  prepared  with 
it  is  of  service  in  the  comedo  stage. 

Eczema  is  produced  by  the  action  of  the  pyogenic  cocci  (M. 
pyogenes,  var.  aureus  and  albus).  Virulent  cultures  of  these  organ- 
isms, with  or  freed  from  their  toxins,  seem,  however,  to  produce 
an  impetigo  rather  than  eczema.  But  the  filtered  cultures,  i.e. 
toxins,  are  harmful  to  the  skin,  and  when  applied  to  it  for  one  or 
two  days  by  means  of  moist  warm  pads  a  typical  papular  or  vesi- 
cular eczema  ensues.  Probably  in  the  human  subject  in  addition 
to  the  micro-organisms  some  peculiarity  in  the  soil  is  necessary 
for  the  disease  to  develop.2  In  so-called  seborrhceic  eczema,  a 
non-liquefying  micrococcus  which  forms  butyric  acid  has  been 
isolated. 

Impetigo. — The  large  vesiculo-bullous  eruption  of  impetigo  con- 
tagiosa  is  caused  by  the  Streptococcus  pyogenes  ;  the  small  pustule 
in  the  neighbourhood  of  hair-follicles,  impetigo  of  Bockhart,  is 
caused  by  the  M.  pyogenes  var.  aureus.  The  B.  diphtheria  may 
also  cause  an  impetigo  (p.  273). 

Pemphigus. — A  diplococcus  has  been  isolated  in  acute  pemphigus 
by  Demme,  and  in  the  chronic  form  by  Dahnhardt.  Bulloch  and 
Russell  Wells,  in  this  country,  seem  to  have  isolated  an  identical 
organism,  and  the  following  description  of  it  is  taken  from  their 
papers.  Cocci  0-8  to  1-5  p  in  diameter,  mostly  arranged  as  diplo- 
cocci,  and  staining  by  Gram's  method.  On  surface  agar  the  organ- 

1  Journ.  of  Pathol.  and  Bacterial.,  vol.  xiv,  1910,  p.  224. 

2  Whitfield,  Practitioner,  February  1904,  p.  202. 


MEASLES  561 

ism  forms  a  thick,  white,  shining  growth.  In  stab  agar  the  growth 
has  a  "  nail-shaped  "  appearance.  The  colonies  on  agar  are  at 
first  round,  but  later,  in  seven  days,  they  throw  out  lateral  pro- 
jections and  assume  a  rosette  appearance.  On  gelatin  the  growth 
is  slow  and  slight,  with  some,  but  not  marked,  liquefaction.  On 
blood-serum  the  growth  resembles  that  on  agar.  On  potato  a 
whitish,  semi-transparent  film  forms.  Milk  is  curdled.  In  brotlj 
it  causes  a  general  turbidity,  with  a  whitish  sediment,  and  some- 
times a  pellicle,  which  soon  sinks.  Guinea-pigs  and  mice  inoculated 
or  vaccinated  with  the  organism  died  in  four  to  eight  days,  fine 
haemorrhage,  occurring  in  the  lungs,  and  the  cocci  being  obtained 
from  the  blood.  No  bullae  appeared  on  the  skin.  The  B.  pyo- 
cyaneus  may  cause  dermatitis  and  bullous  eruptions  (see  p.  239). 

The  pyogenic  cocci  or  their  toxins  may  produce  various  bullous 
eruptions,  e.g.  pemphigus  neonatorum  and  contagiosus  and  hydroa 
gestationis.1 

Herpes  zoster. — Pfeffer  observed  bodies  in  the  cells  of  the  vesicles 
which  he  believed  to  be  protozoa.  Gilchrist,  however,  regards 
these  merely  as  altered  nuclei. 

FOOT  AND  MOUTH  DISEASE. — Various  organisms  have  been 
described  in  this  disease,  but  a  German  commission  comprising 
Loffler  and  Abel  2  stated  that  they  were  unable  to  prove  its  etio- 
logical  significance.  Lofiler  and  Frosch  have  determined  that  the 
organism  must  be  a  very  minute  one,  as  it  passes  through  the 
smallest- pored  porcelain  filter. 

MASTOID  DISEASE. — See  "  Otitis  Media." 

MEASLES. — Doehle  and  Behla  described  small  flagellated  bodies 
which  they  believed  to  be  protozoa  in  this  disease.  Canon  and 
Pielicke  found  small  bacilli  in  the  blood,  which  Tchaikovsky  con- 
firmed. They  are  motile,  do  not  stain  by  Gram's  method,  and 
can  be  cultivated  on  agar  and  serum,  on  which  they  form  delicate 
colonies.  Czajkowski  has  found  a  similar  organism.  Lesage 3 
cultivated  a  small  micrococcus  from  the  nasal  mucus  and  blood, 
which  produced  a  fatal  haemorrhagic  septicaemia  in  animals.  The 
influenza  bacillus  is  present  in  many  cases.  The  organism  is 
probably  a  filter-passer. 

MENINGITIS  may  be  caused  by  S.  pneumonice  (60  per  cent,  of 
acute  cases),  D.  intracellularis,  Still's  diplococcus,  B.  tuberculosis, 
gonococcus,  and  micrococci  and  streptococci. 

MUMPS  (EPIDEMIC  PAROTITIS). — Mecray  and  Walsh  isolated  from 

1  Brit.  Med.  Journ.,  1902,  vol.  i,  p.  73. 

2  Centr.  f.  Bakt.,  xxiii,  1898,  March. 

3  Compt.  Rend.  Soc.  Biol,  1900,  p.  203. 

36 


562  A  MANUAL  OF  BACTERIOLOGY 

the  parotid  and  blood  in  some  cases  of  mumps  a  coccus  resembling 
that  described  by  Laveran  and  Catrin.  It  occurs  chiefly  as  a 
diplococcus,  but  also  in  large  groups.  The  colonies  form  circular, 
white,  shining  points,  with  slow  growth  and  gradual  liquefaction. 
On  potato  a  white  growth  occurs  ;  on  blood-serum  a  plentiful  cream- 
coloured  growth  ;  and  in  litmus  milk  production  of  acid  with 
coagulation. 

NOMA  AND  CANCBUM  OBIS. — Grawitz  in  1890  observed  bacilli 
in  the  affected  tissues  in  this  disease,  others  fusiform  bacilli  with 
or  without  other  organisms  ;  Comba  considered  that  there  was 
probably  no  specific  organism  ;  Durante  found  the  M.  pyogenes, 
Var.  aureus,  with  B.  proteus,  and  Ravenna  the  same  micrococcus 
with  the  typhoid  bacillus.  Diphtheroid  bacilli  have  also  been 
isolated.  Weaver  and  Tunnicliff  l  in  a  case  of  cancrum  oris  observed 
the  presence  of  fusiform  bacilli  and  spirilla.  Hellesen  2  isolated  a 
diplococcus  from  a  case  of  noma.  The  organism  is  not  unlike  the 
pneumococcus,  but  possesses  no  capsule,  is  Gram-positive,  gives 
a  general  turbidity  in  broth  with  acidity,  forms  no  gas  from  glucose, 
curdles  milk  with  acid  production,  and  forms  punctate,  whitish- 
grey,  translucent  colonies  on  surface  agar.  On  inoculation  into 
animals  a  specific  necrosis  was  produced. 

Bishop  and  Ryan,  in  two  out  of  three  cases,  isolated  an  organism 
which  culturally  and  morphologically  resembled  the  diphtheria 
bacillus,  but  which  only  produced  some  local  inflammation  on 
inoculation  into  guinea-pigs.  In  the  third  case  the  M.  pyogenes, 
var.  aureus,  and  the  Streptococcus  pyogenes  were  isolated.  Guizzetti, 
and  Freymuth  and  Petruschky  have  isolated  the  Klebs-LofHer 
bacillus  in  noma. 

OpPLEB-BoAS  BACILLUS. — Met  with  in  the  stomach,  particularly 
in  cases  of  carcinoma,  and  its  detection  is  suggestive  of  this  con- 
dition. The  bacilli  occur  in  masses,  are  long  and  filiform  and  non- 
motile,  and  frequently  join  one  another  at  an  angle.  They  measure 
usually  6-8  p.  in  length,  but  vary  between  3  and  10  p..  The  organism 
has  been  cultivated,  and  is  facultative  anaerobe,  non-sporing  and 
Gram-positive.  It  curdles  milk  and  forms  lactic  acid  from  various 
sugars. 

OTITIS  MEDIA. — The  Streptococcus  pneumonice  is  perhaps  the 
commonest  organism  met  with  ;  next  in  frequency  comes  the 
Streptococcus  pyogenes,  and  then  the  pyogenic  cocci.  In  scarlatinal 
otitis  media,  Blaxall  found  the  S.  pyogenes  to  be  always  present, 
and  generally  accompanied  by  other  organisms,  pyogenic  cocci, 

1  Journ.  Infectious  Diseases,  vol.  iv,  1907,  p.  8  (Bibliog.). 

2  See  Lancet,  1908,  vol.  i,  p.  955. 


PELLAGRA  563 

etc.  In  thirty-seven  cases  of  mastoid  disease  Blake  found  the 
following  organisms,  and  remarks  that  as  a  rule  the  same  were 
found  in  the  middle  ear  : 

Streptococcus       .......  12 

Staphylococcus     .......  5 

Diplococcus  (?  pneumonice)     .....  6 

Streptococcus  and  diplococcus        ....  5 

Streptococcus  and  Bacillus  fetidus  ( ?  colon  bacillus)  3 

Streptococcus  and  Bacillus  pyocyaneus    ...  1 

Streptococcus  and  diplococcus        ....  1 

Streptococcus,  micrococcus,  and  diplococcus    .          .  2 

In  two  of  the  cases  no  organisms  could  be  isolated. 

OZJENA  (ATROPHIC  RHINITIS). — Lowenberg  described  in  this 
disease  encapsuled  bacilli  somewhat  resembling  the  pneumo-bacillus 
morphologically.  Some  Italian  observers  found  bacilli  apparently 
identical  with  the  diphtheria  bacillus.  Abel  *  described  a  bacillus 
somewhat  resembling  the  pneumo-bacillus.  It  is  this  organism 
which  produces  the  atrophy  of  the  mucous  membrane,  but  the 
fetor  is  due  to  the  decomposition  of  the  secretions  produced  by 
other  organisms. 

Perez 2  isolated  an  organism  in  ozsena  (Cocco-bacillus  fetidus 
ozcence)  which  has  the  following  characters :  it  is  a  short  bacillus 
with  rounded  ends,  non-motile,  does  not  stain  by  Gram's  method, 
does  not  liquefy  gelatin,  does  not  ferment  lactose  nor  curdle  milk, 
but  forms  indole  and  ferments  urea.  Its  cultures  are  foul-smelling, 
and  it  is  pathogenic  for  guinea-pigs,  mice,  rabbits,  and  pigeons. 

PELLAGRA. — Many  hypotheses  have  been  propounded  to  account 
for  the  causation  of  this  disease,  in  which  no  micro-organism  has 
been  detected  with  certainty.  It  formerly  was  supposed  to  be  due 
to  the  consumption  of  maize,  which  contains  toxic  substances. 
Lombroso  suggested  that  spoilt  maize  is  the  cause,  toxic  substances 
being  produced  by  Penicillium  glaucum.  Of  parasitic  theories, 
Ceni  and  others  suggest  infection  with  Aspergillus  fumigatus  or 
A.  flavescens.  Tizzoni  attributes  it  to  the  pleomorphic,  Strepto- 
bacillus  pellagrce  (which  may  be  a  pleomorphic  form  of  an 
actinomycotic  organism).  Sambon  on  epidemiological  data 
believes  that  a  protozoan  parasite  is  the  agent  and  is  trans- 
mitted by  small  biting  flies  of  the  genus  Simulium.  The  sun's  rays 
have  also  been  supposed  to  cause  the  affection. 3 

1  Zeitschr.  f.  Hyg.,  xxi,  p.  89. 

2  Ann.  de  Vlnst.  Pasteur,  xiii,  1899,  p.  937,  and  xv,  1901,  p.  409. 

3  See   First   Progress   Rep.    of    the   Thompson-McFadden    Pellagra 
Commission. 


564 


A  MANUAL  OF  BACTERIOLOGY 


T  PERITONITIS. — Treves  gives  the  following  Table  of  the  micro- 
organisms found  in  peritonitis  : 


Frankel 

Tavel  and  Tanz 

Found  alone 

Found  alone 

Found  in 
association 

Bacillus  coli  communis 

11 

15 

16 

Streptococcus 
Staphylococcus 
Pneumococcus 

7 
1 
1 

3 
2 

0 

15 
6 

2 

20 

20 

39 

Dudgeon  1  believes  the  B.  coli  is  frequently  a  secondary  agent  and 
not  the  primary  infection.  He  finds  that  the  M.  pyogenes,  var. 
albus,  is  very  commonly  present  from  the  first,  and  may  exert  a 
protective  action  by  determining  the  occurrence  of  phagocytosis. 

PSILOSIS  OR  SPRUE. — Carnegie  Brown  2  considers  this  disease  to 
be  due  to  an  abnormal  fermentation  in  the  intestine  brought  about 
by  some  organism,  bacterial  or  protozoan,  which  has  not  yet  been 
isolated. 

PUERPERAL  FEVER. — This  condition  may  be  either  a  localised 
infection  with  intoxication  (sapraemia),  or  a  localised  infection  with 
general  infection  (puerperal  septicaemia)  ;  in  both  the  primary 
seat  of  infection  may  be  perinseal  or  vaginal  lacerations,  or  the 
contents  of  the  uterus  or  the  placental  site.  The  infecting  organisms 
may  be  S.  pyogenes,  pure  (20  per  cent.),  or  with  other  organisms 
(30  per  cent.),  occasionally  the  S.  pneumonia?,  B.  coli,  M.  pyogenes, 
var.  albus,  M.  pyogenes,  var.  aureus,  M.  gonorrhoea?,  B.  Welchii, 
and  diphtheroid  bacilli.  These  are  rarely  alone,  but  generally  occur 
with  one  or  other  of  the  organisms  named.  The  B.  diphtheria?  may 
exceptionally  be  met  with.3 

PURPURA. — Hsemorrhagic  septicaemia  may  be  caused  by  a  number 
of  capsulated  bacilli  allied  to  the  B.  pneumonia?  of  Friedlander  * 
(see  pp.  258,  404),  as  well  as  by  streptococci  and  pyogenic  cocci. 
Paratyphoid  infection  may  be  accompanied  with  purpura. 

1  Bacteriology  of  Peritonitis  (Constable,  1905). 

2  Sprue  and  its  Treatment  (Bale,  Sons,  &  Danielsson,  1908). 

3  See  Foulerton,  Practitioner,  March,  1905,  p.  387. 

*  See  Howard,  Journ.  Exp.  Med.,  vol.  iv,  1899,  p.  149  (Bibliog.). 


ACUTE  RHEUMATISM  565 

PYORRHCEA  ALVEOLARIS  (Rigg's  disease). — Goadby1  has  found 
the  following  organisms  to  be  probably  causative  in  this  disease : 
M.  citreus  granulatus,  M.  pyogenes,  var.  aureus,  streptococci,  M. 
catarrhalis,  and  diphtheroid  bacilli,  and  has  used  vaccine  treatment 
with  success.  Eyre  and  Payne  2  have  found  similar  organisms, 

RAT-BITE  DISEASE. — A  disease  occasionally  met  with  in  England 
but  commoner  in  Japan,  and  consequent  on  the  bite  of  a  rat.  It 
is  characterised  by  weekly  bouts  of  severe  fever  lasting  two  or  three 
days.  No  organism  has  been  detected.3 

RHEUMATISM  (ACUTE). — The  opinion  has  gained  ground  of  late 
years  that  acute  rheumatism  is  an  infective  disease.  A  number  of 
observers  have  isolated  streptococci  and  micrococci  in  this  disease, 
and  Singer  regards  the  disease  as  merely  an  attenuated  form  of 
pyaemia.  Menzer  considers  that  rheumatic  fever  is  not  due  to  any 
one  organism,  but  is  a  particular  reaction  in  predisposed  persons 
to  various  microbes,  especially  streptococci.  In  1897  Achalme 
isolated  an  anaerobic  anthrax-like  bacillus  from  several  cases. 
This  bacillus  agrees  in  all  its  characters  with  the  B.  Welchii  (enteri- 
tidis  sporogenes),  and  is  believed  by  the  writer  4  to  be  identical  with 
the  latter  ;  it  is  probably  a  terminal  infection  or  a  contamination. 
Poynton  and  Paine  5  in  1899  obtained  from  eight  successive  cases 
a  diplococcus  (D.  rheumaticus)  which  in  broth  develops  into  a 
streptococcus.  Injected  intravenously  into  rabbits  the  diplococcus 
frequently  produces  enlargement  and  inflammation  of  the  joints 
with  effusion,  and  occasionally  valvulitis  and  endocarditis.  In 
man  the  organism  was  demonstrated  in  the  vegetations,  pericardium, 
tonsils,  and  rheumatic  nodules,  and  has  been  isolated  from  the 
blood,  pericardial  fluid,  cardiac  vegetations,  and  tonsils. 

Andrewes  and  Horder  found  that  two  strains  of  the  D.  rheumaticus 
corresponded  with  the  S.  fcecalis  (p.  234). 

Beattie  6  also  obtained  a  streptococcus  from  the  synovial  mem- 
brane of  cases  of  acute  rheumatism,  which  regularly  produced 
arthritis,  and  occasionally  endocarditis,  in  rabbits.  Goadby  has 
observed  similar  effects  with  a  streptococcus  obtained  from  the 
mouth. 

The  manner  in  which  typical  acute  rheumatism  generally  reacts 

Proc.  Eoy.  Soc.  Med.,  February  1910  (Odontological  Section). 
Ibid.  December  1909. 

See  Hewlett  and  Rodman,  Practitioner,  July  1913,  p.  86. 
Trans.  Path.  Soc.  Lond.,  vol.  lii,  pt.  ii,  1901,  p.  115. 
Lancet,  1900,  vol.  ii,  p.  861  et  seq.  ;  Trans.  Path.  Soc.  Lond.,  vol.  Iv, 
1904,  p.  126. 

6  Journ.  Pathol.  and  Bacteriol.,  vol.  xiv,  1910,  p.  432. 


566  A  MANUAL  OF  BACTERIOLOGY 

to  salicylates  suggests  a  protozoan  organism,  if  an  organism  be  the 
cause. 

RHEUMATOID  ARTHRITIS  (ARTHRITIS  DEFORMANS). — This  disease, 
which  is  probably  not  a  single  one,  may  sometimes  be  caused  by 
an  intestinal,  urinary,  pyorrhceic,  or  other  toxaemia.  Blaxall l 
found  in  the  synovial  fluid,  and  occasionally  in  the  blood,  a  minute 
bacillus  measuring  2  p.  in  length.  It  possessed  marked  polar 
staining,  was  decolorised  by  Gram's  method,  and  could  only  be 
stained  by  prolonged  (3-5  days)  immersion  in  anilin  methylene  blue. 
The  organism  can  be  cultivated  on  agar,  on  serum,  and  in  broth. 
In  a  clear  broth,  after  three  days,  minute  shining,  yellowish  particles 
appear  and  increase  in  amount,  giving  rise  on  shaking  the  flask  to 
an  appearance  of  "  gold  dust."  Inoculation  experiments  on 
animals  failed. 

Poynton  and  Paine  2  isolated  a  diplococcus  (?  a  form  of  their 
D.  rheumaticus)  from  an  osteo-arthritic  joint,  which  produced 
arthritis,  with  osteo-arthritic  changes,  when  injected  intravenously 
into  rabbits. 

Crowe3  has  found  a  micrococcus  of  peculiar  type  in  the  urine 
in  many  cases.  It  may  be  isolated  on  the  neutral-red  egg  medium 
(p.  235),  and  a  vaccine  prepared  with  it  seems  to  be  of  service  in 
treatment.  The  organism  is  allied  to  the  M.  epidermidis  and  has 
been  named  by  Crowe  M .  deformans. 

RHINOSCLEROMA. — A  bacillus  has  been  described  in  this  disease. 
It  is  a  short  rod,  with  rounded  ends,  encapsuled,  and  frequently 
linked  in  pairs.  The  organism  is  non-motile,  does  not  stain  by 
Gram's  method,  and  forms  on  gelatin  a  whitish  growth  without 
liquefaction  like  that  of  Friedlander's  pneumo -bacillus.  Milk  is  not 
coagulated.  The  organism  is  slightly  pathogenic.  It  is  doubtful 
if  it  is  the  causal  agent. 

RINDERPEST. — Simpson,  Koch  and  Eddington  described  bacilli 
in  this  disease,  but  Nicolle  and  Adil-Bey  have  found  that  the  virus 
passes  through  a  procelain  filter,  and  the  organism  therefore  is 
probably  ultra-microscopic. 

TRACHOMA. — Various  organisms  have  been  observed  in  this 
disease,  e.g.  a  diplococcus  by  Sattler,  gonococcal-like  organisms 
by  Lindner  and  others  (it  is  even  suggested  that  the  organism  may 
be  an  "involuted"  gonococcus),  the  Koch-Weeks  bacillus,  the 
Morax-Axenfeld  diplobacillus  and  the  pneumococcus.  Minute 
cell-inclusions,  which  may  be  demonstrated  by  the  Giemsa  method, 

1  Lancet,  1896,  vol.  i,  p.  1120  (Bibliog.). 

2  Brit.  Med.  Journ.,  1902,  vol.  i,  p.  79. 

3  Lancet,  i,  1913,  p.  1377,  and  ii,  1913,  p.  1460. 


UNDULANT  FEVER  567 

are  present  in  the  epithelial  cells,  regarded  by  Halberstaeder  and 
Prowazek  as  Chlamydozoa  *•  (p.  537).  The  disease  is  inoculable 
on  apes  and  the  virus  is  stated  to  be  a  filter-passer.  The  causative 
organism  cannot  yet  be  said  to  be  known. 

UNDULANT  FEVEK.2 — Synonyms  :  Rock,  Mediterranean  or  Malta 
fever.  A  disease  met  with  especially  on  the  Mediterranean  littoral, 
but  also  in  South  Africa,  India,  China,  the  Philippines,  and  the 
subtropical  countries  of  America,  and  clinically  often  simulating 
typhoid  fever. 

A  minute  micrococcus  (M.  melitensis},  first  described  by  Bruce, 
is  the  cause  of  the  disease. 

Microscopically,  the  organism  from  cultures  occurs  as  a  coccus, 
single,  in  pairs,  or  in  short  chains  ;  it  is  easily  stained  by  the  ordinary 
anilin  dyes,  but  is  Gram-negative.  In  hanging-drop  cultures  it 
shows  decided  movement,  which  may  be  only  an  active  Brownian 
movement,  but  is  perhaps  a  true  motility  inasmuch  as  Gordon  has 
described  the  presence  of  flagella  (other  observers  have  failed  to 
find  them).  The  organism  may  be  isolated  from  the  spleen  of  a 
cadaver. 

On  agar  it  grows  as  minute  transparent  colonies,  which  first 
appear  when  inoculated  from  the  spleen  in  90  to  125  hours.  In 
thirty-six  hours  more  the  colonies  become  amber-coloured,  and 
later  still  in  four  to  five  days,  they  become  opaque,  of  a  slightly 
orange  colour,  and  round  with  granular  margins.  On  gelatin  a 
whitish  growth  slowly  forms  without  liquefaction,  and  in  broth  a 
diffused  cloudiness  forms,  with  a  white  deposit  and  without  film- 
formation.  Litmus  milk  becomes  alkaline  without  curdling.  Alkali 
is  also  produced  in  glucose  media,  but  galactose,  maltose,  and 
saccharose  are  unchanged  (see  Table,  p.  248).  The  distribution  of 
the  M .  melitensis  in  the  body  corresponds  closely  with  that  of  the 
B.  typliosus  ;  thus  it  is  abundant  in  the  spleen,  relatively  scanty 
in  the  blood,  and  is  excreted  in  the  urine. 

The  M.  melitensis  maintains  its  vitality  outside  the  body  in 
the  dry  state  in  dust  or  on  clothing  for  two  to  three  months,  in  tap- 
or  sea-water  for  a  month.  The  thermal  death-point  is  about 
55°  C. 

Inoculated  into  animals  no  result  usually  ensues  ;  in  the  monkey, 
however,  a  febrile  condition  is  produced,  with  enlarged  spleen, 
sometimes  terminating  in  death,  the  course  of  the  temperature 
resembling  that  of  the  disease  in  man.  By  intra-cerebral  inoculation 

1  Berl.  Idin.  Woch.  No.  24,  1909. 

2  See  Reports  of  the  Mediterranean  Fever  Commission  (Royal  Society), 
pts.  i-vii,  Harrison  &  Sons,  1904-1907. 


568  A  MANUAL  OF  BACTERIOLOGY 

Durham  found  that  the  organism  becomes  pathogenic  for  the  rabbit 
and  guinea-pig,  otherwise  it  is  without  effect.  For  the  diagnosis 
of  the  disease  the  agglutination  reaction  is  most  valuable.  It  may 
be  carried  out  by  the  microscopic  method,  a  forty-eight-hours' 
broth  culture  being  employed,  the  details  of  the  process  being  the 
•same  as  described  at  p.  191.  Dilutions  of  1  in  30,  1  in  50,  and 
1  in  100  should  be  prepared,  as  well  as  controls  with  normal  serum, 
for  old  laboratory  strains  sometimes  agglutinate  with  normal  serum 
in  dilution  of  1  in  20  or  30  (see  p.  192.  Neglect  of  this  precaution 
ed  Bentley  to  ascribe  kala-azar  to  a  Malta  fever  infection).  The 
organism  being  minute,  it  is  necessary  to  use  the  yL-inch  oil-immer- 
sion, the  £-inch  with  a  high  eyepiece  and  draw-tube  extended,  or 
better,  a  J-inch  dry  objective.  Bassett-Smith  *•  for  agglutination 
tests  prefers  the  sedimentation  method,  for  which  an  emulsion  of 
a  forty-eight-hour  old  agar  culture  in  physiological  salt  solution 
should  be  employed.  Three  dilutions  of  the  serum  are  made, 
1  in  40, 1  in  100,  and  1  in  400,  and  the  tubes  are  placed  in  the  blood- 
heat  incubator  for  two  hours  and  the  results  noted.  The  tubes 
should  then  be  allowed  to  stand  at  laboratory  temperature  and  the 
results  recorded  after  a  further  period  of  twelve  hours.  In  some 
two  thousand  observations,  only  once  was  a  positive  agglutination 
obtained  with  a  control  serum.  Complement-fixation  tests  may 
also  be  employed  and  are  satisfactory.  Absence  of  agglutination 
does  not  necessarily  negative  a  diagnosis  of  undulant  fever  :  in  cases 
of  long  duration  it  may  be  absent.  Isolation  of  the  organism  from 
the  blood  is  another  method  that  may  be  used,  but  similarly  may 
fail  in  long-standing  cases. 

The  disease  may  be  conveyed  to  monkeys  by  contact,  by  inhala- 
tion of  infected  dust,  and  by  feeding.  Mosquitoes  and  other  insects 
do  not  seem  to  convey  it. 

The  investigations  of  the  Mediterranean  Fever  Commission  have 
shown  that  the  main  source  of  infection  of  man  is  by  goat's  milk. 
Goats  may  be  infected  (and  are  largely  so  in  endemic  districts,  e.g. 
Malta  and  South  Africa)  without  showing  any  symptoms,  and 
excrete  the  organism  in  large  numbers  in  their  milk.  Since 
goat's  milk  has  been  boiled  the  incidence  of  the  disease  in  Malta 
has  fallen  from  663  cases  in  1905  to  7  cases  in  1907  in  the  Army, 
and  in  the  Navy  there  were  no  cases  in  1907  (Bruce). 

Toxin,  vaccine,  and  serum  therapy. — The  M.  melitensis  forms  no 
extra-cellular  toxin,  but  Macfadyen  obtained  an  endotoxin  by 
disintegration.  Attempts  to  prepare  an  anti-serum  have  not  been 
successful.  A  vaccine  prepared  with  cultures  killed  by  heat  (see 

1  Journ.  of  Hyg.,  xii,  1912,  p.  497. 


SKIN  AND  CONJUNCTIVA  569 

p.  219)  has  been  used  in  the  chronic  form  of  the  disease  by  Bassett- 
Smith  l  and  others  with  some  amount  of  success  (dose  100  to  500 
millions) 

An  organism,  the  M.  paramelitensis,  has  been  found  by  Negre 
and  Raynaud  in  certain  cases  of  undulant  fever.  In  such  cases, 
the  blood  may  not  agglutinate  the  M.  melitensis  but  does  agglu- 
tinate the  M.  'paramelitensis.  A  case  of  this  kind  is  recorded  by 
Bassett-Smith.2  As  regards  treatment,  yeast  or  yeast-products 
have  been  found  of  service  in  the  neuritis  of  the  disease.  Vaccines 
(100  to  500  millions)  should  be  given  every  five  to  seven  days  :  they 
are  contra-indicated  when  the  pyrexia  is  continuous  or  remittent. 


Micro-Organisms  of  the  Skin  and  Mucous 
Membranes 

Skin. — In  the  normal  clean  skin  micro-organisms  are  scattered 
here  and  there  in  cracks  of  the  horny  layer  and  in  crevices  around 
hairs  and  glands,  but  such  skin  is  not  swarming  with  microbes. 
The  S.  pyogenes  and  M.  pyogenes,  var.  aureus,  albus,  and  citreus, 
and  the  M.  epidermidis  (albus)  of  Welch,  are  the  commonest  (see 
p.  229).  Equally  common  on  the  skin  and  scalp  is  the  scurf  micro- 
coccus  isolated  by  Gordon  (see  Table,  p.  230).  Sarcinae,  bacilli, 
and  moulds  occur  also.  On  the  skin  of  the  groin,  scrotum,  and 
vulva  the  smegma  bacillus  occurs.  From  sweating  feet  various 
organisms  have  been  isolated,  which  on  culture  evolve  a  disagreeable 
odour,  among  which  is  the  Bacterium  fetidum  of  Thin. 

Conjunctive. — Some  observers  have  stated  that  the  conjunctiva 
is  generally  sterile.  A  certain  number  of  organisms  are,  however, 
usually  present,  though  they  are  not  numerous,  and  if  artificially 
inoculated  the  excess  is  rapidly  eliminated.  The  B.  xerosis  can 
often  be  isolated. 

Randolph  3  states  that  the  normal  conjunctiva  always  contains 
organisms,  the  commonest  species  being  the  Micrococcus  epidermidis 
(albus)  of  Welch. 

Lawson  4  found  the  normal  conjunctiva  to  be  sterile  in  20  per  cent, 
of  cases  and  pyogenic  cocci  to  be  rare,  and,  when  present,  non- 
virulent. 

1  Journ.  of  Hygiene,  vol.  vii,  1907,  p.  115. 

2  Journ.  Trop.  Hed.  and  Hygiene,  February  15,  1913. 

3  Archives  of  OphthalmoL,  vol.  xxvi,  1897,  p.  379. 

4  Trans.    Jenner   Inst.    Prev.    Med.,    vol.    ii,    p.    56  ;     also    Griffith, 
Thompson  Yates  Lab.  Rep.,  vol.  iv.  pt.  i,  1901,  p.  99. 


570  A  MANUAL  OF  BACTERIOLOGY 

Nose. — In  the  anterior  nares  crusts  and  vibrissae  micro-organisms 
are  present  in  great  abundance,  but,  contrary  to  the  usual  opinion, 
StClair  Thomson  and  the  writer  l  showed  that  the  mucous  mem- 
brane of  the  interior  of  the  nose  is  comparatively  sterile,  and  when 
organisms  are  present  they  are  very  scanty  compared  with  the 
number  of  organisms  inspired.2  Moreover,  organisms  artificially 
deposited  were  found  to  be  rapidly  disposed  of.  After  two  hours, 
for  example,  prodigiosus  inoculated  on  to  the  inferior  turbinate 
could  not  be  detected  by  cultivation.  Wurtz  and  Lermoyez 
asserted  that  the  nasal  mucus  is  germicidal,  but  StClair  Thomson 
and  the  writer  3  were  unable  to  confirm  this,  though  it  may  have 
an  inhibitory  action. 

Air-passages. — Below  the  larynx  under  normal  conditions  the 
air-passages  are  free  from  micro-organisms.  Expired  air  is  also 
free  from  organisms,  and  the  air  from  the  naso-pharynx  after  passing 
through  the  nasal  cavities  is  deprived  of  the  majority  of  its 
organisms.4 

Mouth. — Micro-organisms  of  all  kinds  are  present  in  the  buccal 
cavity  in  the  greatest  abundance — leptothrix,  bacilli,  pyogenic 
cocci,  sarcinae,  and  spirilla  are  almost  always  to  be  found.  The 
Streptococcus  pyogenes,  M.  pyogenes,  var.  aureus,  and  Streptococcus 
pneumonice  are  frequently  present.  Certain  organisms  have  their 
normal  habitat  in  the  mouth,  are  difficult  to  cultivate,  and  are  of 
considerable  importance  in  the  production  of  dental  caries.5  Well- 
defined  micrococci  and  streptococci  also  occur  in  the  saliva  (M. 
salivarius,  p  231,  and  8.  salivarius,  p.  234).  The  normal  saliva  is 
germicidal  to  some  extent.  (See  also  p.  460.) 

Stomach  and  intestine. — Although  a  vast  number  of  organisms 
gain  access  to  the  stomach,  a  large  number  are  destroyed  by  the 
acid  gastric  juice.  At  the  same  time  a  considerable  proportion 
are  able  to  survive — sarcinae,  and  lactic  and  butyric  acid  bacilli. 
In  normal  nurslings  the  mouth  and  stomach  contain  few  bacteria — 
a  few  cocci,  and  some  bacilli  of  the  B.  coli  and  B.  lactis  aerogenes 
groups.  The  small  intestine  contains  remarkably  few  organisms 
of  the  same  types.  In  the  large  intestine  bacteria  are  extremely 
numerous,  particularly  Gram-positive  ones.  These  are  mostly 

1  Medico-CMrurg.  Trans.,  vol.  Ixxviii,  1895  (Bibliog.). 

2  Other   observers,    however,   have   not    altogether   confirmed   this. 
See  Iglauer,  Laryngoscope,  1901,  November,  p.  363. 

3  "  The  Fate   of  Micro-organisms    in  Inspired  Air,"   Lancet,   1896 
January  11. 

4  Ibid. 

5  See  Goadby,  Mycology  of  the  Mouth. 


STOMACH  AND  INTESTINE  571 

slender,  slightly  curved  bacilli  of  moderate  size,  the  B.  bifidus  of 
Tissier,  which  often  has  a  bifid  extremity,  also  a  somewhat  similar 
organism,  B.  acidophilus  of  Moro,  but  capable  of  developing  in  an 
acid  medium,  a  few  B.  Welchii,  and  a  diplococcus.  The  Gram- 
negative  forms  are  B.  coli,  B.  lactis  aerogenes,  and  cocci.  In  bottle- 
fed  children  the  same  organisms  occur,  but  the  preponderating 
organisms  are  Gram-negative  of  the  B.  coli  type,  with  many  cocci 
and  streptococci.  In  childhood  and  adolescence  organisms  of  the 
bifidus  type  become  less  numerous  but  putrefactive  anaerobes 
become  more  so,  particularly  B.  Welchii  and  B.  putrificus  (coli)  of 
Bienstock  ;  the  latter  is  a  long,  slender,  Gram-positive  bacillus  with 
large  terminal  spores.  During  adult  life  the  putrefactive  anaerobes 
tend  to  become  still  more  numerous,  and  the  putrefactive  decom- 
positions they  produce  are  regarded  by  Metchnikofi  as  standing  in 
causal  relation  to  old  age.  In  the  healthy  adult  the  stomach, 
duodenum  and  jejunum  contain  relatively  few  organisms,  from 
the  lower  ileum  to  the  rectum  the  intestinal  contents  are  crowded 
with  bacteria,  and  the  greatest  number  of  anaerobic  organisms  occur 
here  and  putrefactive  changes  are  most  in  evidence.1  Kendall2 
has  described  the  presence  of  a  bacillus  (B.  infantilis)  in  large 
numbers  in  a  condition  of  infantilism,  associated,  according  to 
Herter,  with  chronic  intestinal  infection.  The  organism  is  a  Gram- 
positive,  motile,  sporing  bacillus  belonging  to  the  subtilis  group. 
It  is  aerobic  and  facultatively  anaerobic,  grows  readily  on  the 
ordinary  culture  media,  and  ferments  dextrose  and  saccharose  with 
the  production  of  acid  only,  but  lactose  is  hardly  attacked.  In  a 
dog  and  a  monkey  diarrhoea  was  produced  by  feeding  with  it. 

Urinary  and  genital  organs. — The  meatus  urinarius  and  distal 
portion  of  the  urethra  contain  a  few  organisms,  which  increase  in 
number  in  inflammatory  conditions,  and  Gram-negative  cocci  may 
be  found  (see  p.  248).  The  deeper  portion  of  the  urethra,  however, 
is  free  from  organisms,  and  the  bladder  is  sterile.  The  genital 
tract  in  the  female  up  to  the  middle  zone  of  the  cervix  contains 
organisms,  but  the  uterus  and  Fallopian  tubes  are  normally  sterile. 
The  B.  vagince  of  Doderlein,  a  large  Gram-positive  bacillus  capable 
of  growing  in  an  acid  medium,  is  frequently  present  in  considerable 
numbers  in  the  vagina. 

1  See  Herter,  Bacterial  Infections  of  the  Digestive  Tract,  1907. 

2  Journ.  Biolog.  Chemistry,  vol.  v,  p.  419. 


CHAPTER  XXI 

THE  BACTERIOLOGY  OF  WATER,  AIR,  AND  SOIL,  AND 
THEIR  BACTERIOLOGICAL  EXAMINATION— SEWAGE 
—BACTERIOLOGY  OF  MILK  AND  FOODS 

Some  of  the  Commoner  Organisms  found  in  the  Air,  Water 
and  Soil. 

Bacterial  Content  of  Waters  and  the  Factors 
influencing  it.     Filtration,  etc. 

THE  bacterial  flora  of  natural  waters  is  a  very  varied  one. 
The  organisms  met  with  in  surface  waters,  such  as  streams, 
ponds,  and  shallow  wells,  are  derived  from  the  air  and 
soil  through  which  the  water  has  passed,  and  if  not  con- 
taminated from  human  or  animal  sources,  from  the  air  of 
towns,  from  sewage  or  manure,  consist  mainly  of  non- 
pathogenic  bacilli,  the  majority  of  which  are  chromogenic 
and  non-liquefying,  and  develop  best  on  culture  media 
at  a  temperature  of  18°  to  22°  C.  or  thereabouts,  not  at 
blood  heat ;  also  of  some  sarcinse  and  a  few  micrococci ; 
B.  coli  and  B.  Welchii  are  usually  absent.  When,  however, 
the  water  passes  through  cultivated  lands,  or  receives 
sewage,  the  number  of  organisms  is  enormously  increased  ; 
a  large  proportion  of  them  liquefies  gelatin  and  develops 
at  blood  heat,  and  B.  coli  and  B.  Welchii  appear  more  or 
less  numerously.  Whereas  water  from  shallow  wells  has 
a  bacterial  content  nearly  as  great  as  the  surrounding 
surface  water,  that  from  deep  wells,  especially  in  the  chalk, 
is  remarkably  free  from  organisms.  The  following  Table 

572 


BACTERIAL  CONTENT  OF  WATERS          573 

illustrates  the  number  of  organisms  that  may  be  met  with 
in  water  from  different  sources  : 

Source  Number  of  organisms 

per  cubic  centimetre. 

Freshly  fallen  snow    .          .          .  34-38 

Ice (very  variable)  30-1700 

Rain  water  (Paris)      .          .          .  4-5 
Rhone,  above  Lyons            .          .  75 
Rhone,  below  Lyons            .          .  800 
Rhine,  at  Miihlheim  .          .          .  average  about  20,000 
Thames,    at    Hampton    (Frank- 
land)     (variable)  2000-90,000 

Deep   well  in   the   chalk   (Kent 

Company)      ....  3-19 
Surface  well       ....  1200 
Spring    water,    Reigate    (Frank- 
land)     8 

Lake  of  Lucerne         .          .          .  8-50 

Loch  Katrine  (Frankland)            .  74 
Filtered  water  supplied  to  London 

(Houston)       ....  average  rarely  exceeds  100 

Sewage  (Frankland)             .          .  26,000,000 

The  number  of  bacteria  in  a  natural  water  varies  con- 
siderably with  its  source,  at  different  seasons,  and  under 
different  climatic  conditions.  The  Table x  on  p.  575 
illustrates  the  seasonal  variation  in  certain  raw  London 
waters. 

The  following  factors  modify  the  number  of  organisms 
present  in  the  water  : 

(1)  Storage  of  unfiltered  water. — A  large  storage  capacity 
permits  of  the  water  being  admitted  when  the  source 
(river,  etc.)  is  in  its  best  condition,  so  that  foul  water,  in 
flood  time  or  drought,  may  be  avoided.  Moreover,  storage 
alone  usually  markedly  diminishes  the  number  of  organisms, 
partly  by  subsidence,  partly  by  lack  of  aeration,  and  partly 
probably  owing  to  the  struggle  for  existence  going  on 
among  them  (see  also  p.  361). 

1  Houston,  Seventh  Ann.  Rep.  Hetropol.  Water  Board,  1913. 


574  A  MANUAL  OF  BACTERIOLOGY 

(2)  Thickness  of  fine  sand  in  the  filter-beds. — Efficient 
sand  nitration  removes  quite  99  per  cent,  of  the  organisms 
originally  present.     The  fine  sand  only  has  to  be  taken 
into  account  in  estimating  the  removal  of  organisms  and 
efficiency  of  a  filter  bacteriologically.     It  probably  should 
form  a  layer  not  less  than  3  ft.  to  3  ft.  6  in.  in  thickness. 
Moreover,  a  filter-bed  is  not  efficient  at  first,  but  becomes 
so  when  the  surface  film  forms,  composed  of  sedimented 
particulate  matter,  and  of  a  zoogloeal  mass  of  bacteria  and 
algae. 

(3)  The  rate  of  filtration. — The  removal  of  organisms  is 
less  perfect  when  the  rate  of  filtration  is  increased  ;    this 
should  not  exceed  about  1*5  gallons  per  square  foot  per 
hour. 

(4)  The   renewal   of  the   filter-beds. — New,    or   recently 
cleaned,  filter-beds  allow  a  greater  number  of  organisms 
to  pass  through.     The  beds  must  be  cleaned  from  time 
to  time  by  raking  up  and  clearing  away  the  surface  layer 
of  sand,  for  as  time  goes  on  the  rate  of  filtration  becomes 
slower  and  slower,  though  the  bacterial  efficiency  of  the 
filter-beds  does  not  appear  to  be  reduced  by  prolonged 
use.       The     normal    bacterial    efficiency    seems    to    be 
rapidly   regained    after    cleaning — within    two    or    three 
days. 

Besides  storage  and  filtration,  sedimentation  in  the 
presence  of  fine  particles,  either  naturally  present  or 
artificially  added,  may  also  effect  a  marked  removal  of 
micro-organisms  from  water.  Thus,  by  the  addition  of 
alum,  an  old  method  of  clarifying  turbid  water,  a  large 
number  of  the  organisms  present  are  carried  down  in  the 
precipitate. 

The  Clark  process  of  softening  water  may  also  reduce 
the  number  of  organisms  present,  but  is  very  uncertain 
(Moor  and  Hewlett).  By  the  Porter-Clark  rapid  process, 
however,  in  which  the  precipitate  of  calcium  carbonate  is 


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576  A  MANUAL  OF  BACTERIOLOGY 

removed  by  filtration  through  canvas  bags,  very  con- 
siderable purification  is  effected.1 

Houston  has  introduced  an  "  excess  lime "  method. 
Enough  lime  is  added  to  the  water  to  render  it  decidedly 
alkaline  and  germicidal  for  the  colon  bacillus  in  five  to 
twenty-four  hours  (for  raw  Thames  water,  about  1  of  lime 
in  5000  of  water).  At  the  end  of  this  period  a  sufficiency 
of  pure  stored  water  is  added  so  as  to  precipitate  the 
excess  of  lime.  With  Thames  water,  3  parts  of  raw  water 
with  1  part  of  stored  water  would  be  the  approximate 
quantities. 

The  Tables  on  pp.  577  and  578  illustrate  the  influence 
of  storage  and  of  sand  filtration  on  the  bacterial  content 
of  a  water. 

The  Bacteriological  Examination  of  Water2 

The  bacteriological  analysis  of  water  affords  valuable 
indications  as  to  the  purity  or  otherwise  of  a  water,  and, 
if  properly  carried  out,  will  indicate  a  pollution  so  small 
in  amount  as  to  be  incapable  of  detection  by  chemical 
methods. 

The  specimen  of  water  should  be  collected  in  clean  bottles 
of  about  100-200  c.c.  capacity,  sterilised  preferably  by 
heat.  If,  however,  the  bottles  be  thoroughly  cleaned  and 
rinsed  out  with  a  little  strong  sulphuric  acid,  and  then 
thoroughly  rinsed  several  times  with  the  water  to  be 
examined  before  taking  the  specimen,  no  error  will  be 
introduced.  The  stopper  of  the  bottle  should  be  tied 
down  with  a  thin  layer  of  cotton-wool  enclosed  between 

1  Nankivell,  Journ.  of  Hyg.,  xi,  1911,  p.  246  ;  Hewlett  and  Nankivell, 
Rep.  Med.  Off.  Loc.  Gov.  for  1911-12,  p.  350. 

2  See  Savage,  Bacteriological  Examination  of  Water  Supplies  (Lewis, 
1906) ;    Thresh,  Examination  of  Water  and  Water  Supplies  (Churchill, 
Ed.  2,  1913)  ;  Houston,  Gordon  and  others  in  Reps.  Med.  Off.  Loc.  Gov. 
Board,  1899-1904  ;    Houston,  Reports  to  the  Metropolitan  Water  Board 
and  Studies  in  Water  Supply  (Macmillan  &  Co.,  1913). 


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EXAMINATION  OF  WATER  579 

two  pieces  of  muslin,  and  the  bottle  should  be  not  quite 
filled.  In  taking  the  specimen  the  following  details  should 
be  attended  to  : 

(1)  If  taken  from  a  tap,  the  water  should  be  allowed 
to  flow  for  at  least  five  minutes  before  the  specimen  is 
collected. 

(2)  The  water  from  a  cistern  is  not  a  representative 
sample  of  the  water-supply  ;  to  be  so  the  specimen  should 
be  taken  direct  from  the  mains. 

(3)  If  taken  from  a  stream  or  pond,  the  bottle  should  be 
held  about  a  foot  below  the  surface  and  away  from  the 
edge  before  the  stopper  is  removed. 

(4)  If  taken  from  a  well  the  conditions  should  be  noted, 
e.g.  whether  the  well  has  been  recently  disturbed  or  not, 
whether  the  pumps  have  been  in  operation,  etc.,  for  such 
may  markedly  influence  the  number  of  bacteria  found. 

The  specimen  should  then  be  examined  with  as  little 
delay  as  possible,  for  if  allowed  to  stand  for  any  time  a 
large  increase  in  the  number  of  bacteria  may  take  place. 
Frankland,  for  example,  found  that  in  distilled  water, 
even  at  the  ordinary  temperature,  organisms  multiply 
enormously  : 

Number  of  organisms 
Hours  in  1  c.c. 

0 1,073 

6 6,028 

24 7,262 

48 48,100 

In  water  of  good  quality  the  organisms  are  found  to 
multiply  much  more  rapidly  during  the  first  few  days, 
after  which  time  they  become  less  and  less  numerous ; 
but  in  impure  water  multiplication  is  slower,  and  the 
number  more  persistent,  while  in  very  impure  water  the 
number  may  diminish.  It  is  essential,  therefore,  if  reliable 
results  are  to  be  obtained,  for  the  specimen  to  be  examined 


580  A  MANUAL  OF  BACTERIOLOGY 

at  once  (within  three  hours).  If  this  cannot  be  done  the 
specimen  should  be  packed  in  ice ;  the  cold  will  then 
inhibit  multiplication  to  any  extent.  Special  double- 
chambered  metal  boxes  are  made  for  this  purpose  :  the 
bottle  containing  the  sample  (not  less  than  60  c.c.  ;  the 
writer  prefers  to  have  not  less  than  200  c.c.)  is  placed  in 
the  inner  chamber,  the  outer  chamber  (which  surrounds 
the  inner)  being  filled  with  a  mixture  of  ice  and  sawdust, 
and  the  whole  is  packed  in  a  wooden  box  with  felt  lining. 
According  to  Remlingler,1  the  addition  of  10  per  cent,  of 
common  salt  to  the  sample  preserves  the  original  bacterial 
content  of  the  water  unaltered  up  to  ninety-six  hours  after 
taking  the  sample,  without  icing.  Besides  the  sample 
packed  in  ice,  a  "  Winchester  quart  "  of  the  water  may 
also  be  collected  for  examination  for  the  spores  of  the 
B.  Welchii  (enteritidis  sporogenes). 

The  routine  bacteriological  examination  of  the  specimen 
may  be  carried  out  according  to  the  scheme  (here  somewhat 
modified)  drawn  up  by  a  committee  of  the  Royal  Institute 
of  Public  Health.2 

PROCEDURES. — The  following  procedures  should  be 
carried  out : 

(a)  Enumeration  of  the  organisms  which  will  develop 
aerobically  in  gelatin  at  20°  C. 

(b)  Enumeration  of  the  organisms  which  will  develop 
aerobically  in  agar  at  37°  C.     (Enumeration  is  carried  out 
by  counting  the  number  of  colonies  which  develop  in  the 
plates  [see  p.  79].) 

(c)  Search    for    Bacillus    coli,    and    identification    and 
enumeration  of  this  organism  if  present. 

(d)  Search  for,  and  enumeration  of,  streptococci. 

As  a  routine  measure  it  is  not  necessary  to  search  for 

1  Comp.  Rend.  Soc.  BioL,  Ixx,  p.  64. 

2  Journ.  State  Med.,  vol.  xii,  1904,  p.  471. 


EXAMINATION  OF  WATER  581 

the  Bacillus  Welchii  (enteritidis  sporogenes),  but  in  special 
instances  it  may  be  desirable  to  do  so. 

The  bottle  must  be  well  shaken  to  mix  the  sample. 
Before  removing  the  stopper,  it  and  the  neck  of  the  bottle 
should  be  swabbed  with  absolute  alcohol,  which  is  then 
carefully  ignited  and  allowed  to  burn  away. 

MEDIA,  TIME  OF  INCUBATION,  ETC. — For  the  gelatin 
count  ordinary  nutrient  gelatin  is  employed,  the  period  of 
incubation  being  seventy-two  hours.  In  hot  weather  it 
may  be  necessary  to  use  15-20  per  cent,  gelatin  (unless 
an  incubator  which  can  be  cooled  is  available),  but  the 
development  of  the  colonies  is  slower.  For  the  agar  count 
ordinary  nutrient  agar  is  used,  the  period  of  incubation 
being  forty  to  forty-eight  hours. 

The  media  should  preferably  be  recently  prepared  and 
be  standardised  to  a  reaction  of  +10. 

In  addition  to  the  actual  numbers  of  organisms  which 
develop  in  the  gelatin  and  in  the  agar,  a  comparison  of  the 
ratio  of  the  number  of  organisms  developing  in  gelatin  at 
20°  C.  to  those  developing  in  agar  at  37°  C.  also  gives 
useful  indications.  With  a  pure  water  this  ratio  is  gene- 
rally considerably  higher  than  10  to  1  ;  with  a  polluted 
water  this  ratio  is  approached,  and  frequently  becomes 
10  to  2,  10  to  3,  or  even  less.  The  actual  number  of 
organisms  growing  at  blood-heat  is  of  considerable  value 
apart  from  any  question  of  ratio. 

In  certain  instances  it  is  true  that  this  ratio  may  be 
unreliable.  Thus  with  surface  waters,  especially  in  the 
tropics  (as  pointed  out  by  Horrocks)  varieties  of  the 
B.  fluorescens  liquefaciens  and  non-liquefaciens  and  B. 
liquefaciens  may  be  abundant  and  grow  well  at  blood-heat. 

Distilled  water  gelatin  and  agar  have  also  been  recom- 
mended, but  since  the  organisms  of  polluted  water  develop 
better  in  the  ordinary  nutrient  media,  the  latter  are 
preferable  for  routine  use. 


582  A  MANUAL  OF  BACTERIOLOGY 

AMOUNTS  TO  BE  PLATED,  SIZE  OF  DISHES,  etc.  Gelatin.— 
For  an  ordinary  water  amounts  of  Ol,  0*2  and  O3  c.c. 
may  be  plated  in  Petri  dishes  of  about  10  cm.  diameter, 
preferably  done  in  duplicate. 

Agar. — Two  plates  may  be  made  with  0*1  and  O2- 
O3  c.c.,  and  are  preferably  duplicated. 

The  desired  volume  of  water  should  be  run  into  the  sterile  Petri 
dish  by  means  of  a  sterile  1  c.c.  pipette  graduated  in  hundredths, 
The  tubes  of  gelatin  should  be  melted  in  a  water-bath  at  a  low 
temperature  (40°  C.).  A  tube  is  taken  from  the  water-bath,  wiped 
to  prevent  the  adherent  water  running  down  into  the  Petri  dish, 
its  mouth  is  singed  in  the  Bunsen  flame  to  sterilise  it,  and  the 
contents  are  then  quickly  poured  into  the  dish  and  mixed  with  the 
water  by  tilting  the  dish  several  times. 

The  agar  tubes  must  first  be  boiled,  then  cooled  to  about  45°  C., 
and  similarly  treated,  or  surface  plates  may  be  made. 

If  waters  are  constantly  being  examined,  it  saves  trouble  to  have 
the  gelatin  and  agar  in  small  flasks,  30-60  c.c.  of  the  former  and 
20-40  c.c.  of  the  latter  ;  a  flask  of  each  will  then  be  used  for  an 
examination. 

In  dealing  with  an  unknown  water,  and  in  all  cases  of 
doubt,  additional  plates  should  be  prepared  with  a  dilution 
of  the  water  (made  with  sterilised  tap-water)  of  ten  or 
hundred  fold,  according  to  circumstances. 

The  amount  of  the  medium  in  a  plate  should  be  10  c.c. 

The  counting  is  done  with  the  naked  eye,  preferably  in 
daylight,  any  doubtful  colony  being  determined  with  the 
aid  of  a  lens  or  low  power  objective.  The  number  of 
liquefying  colonies  in  the  gelatin  plates  should  also  be 
noted.  The  plates  should  be  inspected  daily,  in  order 
that  the  count  may  be  made  earlier  should  liquefaction 
render  this  necessary. 

In  examining  an  ordinary  drinking-water  there  is  no  need  ever 
to  dilute.  As  1000  or  1500  colonies  can  be  counted  in  a  plate,  and 
if  the  number  on  a  plate  should  be,  owing  to  crowding,  uncountable, 
ipso  facto  this  would  be  sufficient  to  condemn  without  an  actual 
count.  Dilution  is  necessary  when  dealing  with  river  or  other 


EXAMINATION  OF  WATER  583 

water  known  to  be  polluted,  and  of  which  an  estimate  of  the  number 
of  organisms  present  is  desired.  In  order  to  count  the  colonies  if 
very  numerous,  ink  lines  may  be  drawn  across  the  bottom  of  the 
Petri  dishes  so  as  to  divide  them  into  sectors.  Ruled  paper  discs 
(Pakes's  discs)  upon  which  the  dishes  are  placed  can  also  be  obtained. 
The  colonies  in  the  sectors  are  then  much  more  easily  counted : 
or  if  the  colonies  be  very  numerous  and  evenly  distributed,  the 
number  in  two  or  three  of  the  sectors  may  be  counted,  and  the 
total  number  on  the  plate  estimated  by  calculation. 

SEARCH  FOR  BACILLUS  COLI,  ETC. — Various  media  may 
be  employed  for  the  detection,  isolation,  and  enumeration 
of  B.  coli.  The  writer  generally  employs  as  a  preliminary, 
glucose  bile-salt  peptone-water,  but  many  other  media 
may  be  employed,  e.g.  formate  or  neutral-red  broth,  or 
if  the  organism  is  abundant,  neutral-red  bile-salt  agar. 

As  a  routine,  50  c.c.  should  be  the  minimal  quantity 
examined  for  the  presence  of  the  Bacillus  coli,  quantities 
from  a  minimum  of  0*1  c.c.  to  a  maximum  of  50  c.c.  being 
added  to  the  tubes  of  culture  media. 

It  is  preferable  to  add  the  water  directly  to  the  tubes 
of  culture  medium,  even  with  the  larger  amounts,  and 
not  to  concentrate  the  bacteria  by  any  method.  The 
culture  media  may  be  diluted  with  at  least  an  equal  volume 
of  the  water  without  interfering  with  their  cultural  pro- 
perties, and  large  tubes  or  small  flasks  are  used  for  the 
larger  amounts. 

In  the  case  of  glucose  or  lactose  bile-salt  peptone- water, 
the  medium  may  for  the  larger  amounts  be  prepared  of 
double  strength.  The  glucose  or  lactose  bile-salt  peptone 
water  should  be  incubated  at  42°  C.  for  not  less  than 
forty-eight  hours. 

For  composition  of  glucose  formate  broth,  glucose  and  lactose 
bile-salt  media,  and  neutral-red  broth,  see  p.  590,  et  seq.  While  a 
lactose  medium  has  the  advantage  of  excluding  a  number  of  forms 
which,  though  fermenting  glucose,  do  not  ferment  lactose,  and  are 
therefore  not  typical  B  coli,  Houston  has  found  that  a  glucose 


584  A  MANUAL  OF  BACTERIOLOGY 

medium  is  more  delicate  than  a  lactose  one.  For  general  purposes, 
quantities  of  from  0-1  to  25-0  c.c.  may  be  added  to  tubes  of  the 
medium  selected.  For  the  examination  of  an  ordinary  drinking- 
water,  the  writer  usually  employs  five  tubes  with  1  c.c.  of  the 
water  in  each,  two  tubes  (double  strength)  with  10  c.c.  in  each,  and 
one  tube  (double  strength)  with  25  c.c.  For  the  larger  amounts 
large  test-tubes  and  boiling  tubes  must  be  employed. 

If  the  medium  shows  changes  (acid  +  gas)  suggestive  of  the 
presence  of  B.  coli,  it  is  only  presumptive  evidence  of  the  presence 
of  this  organism.  Occasionally  other  organisms  produce  a  similar 
change,  e.g.  B.  lactis  aerogenes,  B.  cloacae.  Hence  the  necessity  for 
the  isolation  and  identification  of  the  organism  as  recommended 
in  the  next  section. 

ISOLATION  OF  BACILLUS  COLI,  IF  PRESENT. — If  indica- 
tions of  the  presence  of  the  Bacillus  coli  be  obtained  in 
the  preliminary  cultivations  (acid  +  gas),  the  organism 
must  be  isolated  and  identified.  If  several  tubes  show 
acid  +  gas,  one  or  two  of  the  tubes  with  the  smallest 
quantities  of  the  water  should  be  used  for  this  purpose. 

This  may  be  done  by  making  surface  cultures  on  plates 
(sloping  tubes  generally  suffice)  of  either  (a)  litmus  lactose 
agar,  reaction  +  10  ;  (6)  litmus  lactose  bile-salt  agar ; 
(c)  Conradi  and  Drigalski  agar,  which  the  writer  generally 
employs ;  or  (d)  ordinary  nutrient  gelatin.  Agar  media, 
incubated  at  37°  C.,  have  the  advantage  of  saving  time. 
(For  composition  of  media,  see  p.  590,  et  seq.) 

IDENTIFICATION  OF,  AND  TESTS  FOR,  THE  BACILLUS 
COLI. — Having  obtained  coli-like  colonies  on  the  plates 
made  from  the  preliminary  cultivations  of  the  water, 
various  tests  must  be  used  for  identification.  The  organ- 
ism should  conform  in  morphology,  motility  and  staining 
reactions  with  the  characters  of  the  typical  B.  coli  as  given 
at  pp.  379-387,  and  must  be  subjected  to  various  cultural 
tests,  e.g.  the  "  flaginac  "  reactions  of  Houston  (p.  384). 
The  writer  generally  employs  these,  with  the  addition  of 
the  fermentation  reactions  given  by  dulcitol,  mannitol, 
and  adonit  litmus  peptone  water,  and  gelatin  for  absence 


EXAMINATION  OF  WATER  585 

of  liquefaction.  If  atypical  Bacilli  coli  (see  pp.  388  and 
389)  are  met  with,  the  fact  should  be  noted,  but  their 
significance  is  not  yet  fully  determined. 

STREPTOCOCCI. — It  is  a  distinct  advantage  to  search 
for  streptococci.  They  may  be  looked  for  by  making 
hanging- drop  preparations  of  the  fluid  media  employed  for 
the  preliminary  cultivation  of  the  B.  coli  (glucose  or  lactose 
bile-salt  peptone  water,  etc.)  The  presence  or  absence  of 
streptococci  in  these  tubes  gives  also  a  quantitative  value 
to  the  examination,  just  as  in  the  case  of  B.  coli,  and  the 
result  obtained  should  be  stated.  The  streptococci  can 
be  readily  isolated  on  Conradi-agar  plates. 

According  to  Houston  (loc.  cit.),  faeces  contain  at  least  100,000 
streptococci  per  gramme.  The  type  of  streptococcus  generally  present 
is  one  forming  short  chains,  producing  a  uniform  turbidity  in 
broth,  acid  and  clot  in  litmus  milk  within  five  days  at  37°  C.,  and 
non-pathogenic  for  mice.  (See  Table,  p.  235.) 

BACILLUS  WELCHII. — As  already  stated,  it  is  not  essential 
as  a  routine  procedure  to  search  for  the  Bacillus  Welchii 
(enteritidis  sporogenes),  though  in  certain  instances  it  may 
be  of  advantage  to  do  so.  A  negative  result  in  such 
cases  is  probably  of  more  value  than  a  positive  one. 

For  the  isolation  of  B.  Welchii,  500  c.c.  of  the  water  may  be 
filtered  through  a  Pasteur-Chamberland  filter,  the  deposit  is  sus- 
pended in  5  to  6  c.c.  of  sterile  water,  and  1  c.c.  of  the  suspension 
added  to  each  of  five  to  six  tubes  of  sterile  milk,  which  are  then 
heated  to  80°  C.  for  ten  minutes  in  a  water-bath,  and  incubated 
anaerobically  at  37°C.  for  forty-eight  hours  (filter-brushing  method). 
A  better  method  *•  is  to  employ  large  boiling  tubes  or  small  Erlen- 
meyer  flasks,  each  containing  25  to  50  c.c.  of  sterile  milk.  To  each 
tube  a  quantity  of  water  equal  to  that  of  the  milk  is  added,  the 
tubes  are  then  heated  in  a  water-bath  to  80°  C.  for  fifteen  to  twenty 
minutes,  some  sterilised  oil  or  melted  vaseline  is  poured  on  the 
surface  to  exclude  air,  the  tubes  are  cooled  in  water  to  37°  C.  or 
thereabouts,  and  incubated  for  forty-eight  hours  at  37°  C.  Not 
less  than  200  c.c.  of  the  water  should  be  used.  The  typical  change 

1  R.  T.  Hewlett,  Trans.  Path.  Soc.  Lond.,  vol.  Iv,  1904,  p.  123 


586  A  MANUAL  OF  BACTERIOLOGY 

in  the  milk  (see  p.  428)  indicates  the  probable  presence  of  the 
organism.  To  make  sure  that  the  change  is  due  to  the  B.  Welchii 
and  not  to  the  C.  butyricum,  1  c.c.  of  the  whey  per  100  grm.  of 
body-weight  should  kill  a  guinea-pig  in  forty  hours  when  injected 
subcutaneously. 

The  virulence  of  a  peptone-water  culture  has  been  suggested  as  an 
index  of  contamination,  but  in  the  writer's  hands  has  not  given 
reliable  results.  If  sufficient  peptone  and  salt  be  added  to  a 
measured  volume  of  the  water  to  form  a  1  per  cent,  solution  of  the 
former  and  a  \  per  cent,  solution  of  the  latter,  the  mixture  incubated 
at  37°  C.  for  twenty -four  hours  and  injected  intraperitoneally  into 
a  guinea-pig,  a  bad  water  is  stated  to  kill,  whereas  a  good  one  does 
not.  The  amount  to  be  injected  is  2  c.c.  and  death  should  ensue 
within  forty -eight  hours. 

INTERPRETATION  OF  RESULTS. — The  interpretation  of 
the  results  of  the  bacterioscopic  examination  of  water  is  a 
difficult  matter,  for  which  experience  is  necessary.  Just 
as  in  chemical  analysis,  it  is  not  possible  to  lay  down  an 
absolute  standard,  a  knowledge  of  the  source  and  sur- 
rounding conditions  being  of  the  greatest  importance  in 
forming  an  opinion.  The  ultimate  aim  is,  of  course,  the 
detection  of  sewage  or  fsecal  pollution  ;  the  bacterioscopic 
analysis  does  not  give  any  information  as  to  the  suitability 
of  the  water  for  household,  trade,  or  factory  purposes. 

Number  of  colonies  on  the  gelatin  plates. — The  number  of 
colonies  represents  approximately  the  number  of  organisms 
in  the  original  sample  capable  of  development  aerobically 
at  20°  C.  in  gelatin.  This  number  in  a  good  water  rarely 
exceeds  100  or  150  ;  in  pure  waters,  particularly  those 
coming  from  deep  chalk-wells,  there  may  be  only  a  few— 
5  to  10  per  c.c.  (the  results  are  always  expressed  in  numbers 
per  cubic  centimetre  of  the  original  water).  In  waters  of 
poorer  quality  the  number  may  approach  500  per  c.c. 
Anything  over  this  casts  suspicion  on  the  water,  and 
1000  per  c.c.  or  more  should  probably  condemn  the  sample, 
always  supposing,  of  course,  that  multiplication  in  vitro 
can  be  excluded  by  the  proper  storage  of  the  sample 


EXAMINATION  OF  WATER  587 

bottle  in  ice.  As  a  rule  in  water  of  good  quality  liquefying 
organisms  are  scanty,  while  in  a  polluted  water  they  are 
numerous. 

Number  of  colonies  on  the  agar  plates. — As  mentioned 
before  (see  p.  581),  it  is  the  ratio  of  the  number  of  organisms 
developing  on  the  agar  plates  to  the  number  of  those 
developing  on  the  gelatin  plates  that  is  of  importance. 

Number  of  B.  coli. — The  detection  and  enumeration  of 
B.  coli  are  regarded  by  all  as  perhaps  the  most  important 
part  of  water  examination.  The  number  of  B.  coli  is  esti- 
mated from  the  amounts  of  water  that  have  been  added 
to  the  tubes  of  media,  which,  however,  assumes  that  the 
organism  is  regularly  distributed  throughout  the  sample, 
and  this  must  so  far  as  possible  be  ensured  by  thorough 
mixing.  The  results  generally  come  out  fairly  concor- 
dantly,  though  irregularities  exceptionally  occur  which 
can  only  be  obviated  by  making  duplicate  sets  of  cultures. 

It  is  better  to  state  the  result  as  "  B.  coli  present  in 

c.c.  of  water  "  rather  than  to  say  that  so  many  B.  coli  are 
present,  though  as  a  matter  of  fact  the  latter  statement 
is  approximately  correct.  Adopting  the  writer's  method 
for  B.  coli  (p.  584),  if  none  of  the  tubes  contains  B.  coli, 
we  say  that  "  B.  coli  is  absent  from  50  c.c.  "  ;  if  the  25  c.c. 
tube  contains  B.  coli,  but  not  the  remainder,  "  B.  coli  is 
present  in  25  c.c.  but  not  in  less,"  and  so  on. 

If  nothing  is  known  about  the  water,  the  following 
standards  may  be  adopted  : 

(a)  Waters  of  good  quality. — B.  coli  absent  in  50  c.c. 
of  the  water. 

(b)  Waters  of  medium  quality. — B.  coli  present  in  50  c.c. 
but  absent  in  25  c.c. 

(c)  Waters  of  poor  quality. — B.  coli  present  in  50  c.c.  and 
25  c.c.,  but  absent  in  10  c.c. 

(d)  Waters    of  suspicious    quality. — B.    coli   present   in 
50  c.c.,  25  c.c.,  and  10  c.c.,  but  absent  in  1  c.c. 


588  A  MANUAL  OF  BACTERIOLOGY 

(e)  Waters  unfit  for  drinking. — B.  coli  present  in  1  c.c. 
or  less. 

Waters  which  show  no  B.  coli  in  50  c.c.  are  of  a  high  degree  of 
purity,  and  therefore  the  proved  absence  of  this  organism  in  this 
amount,  and  still  better  in  larger  quantities,  is  of  great  value. 

B.  coli  should  be  absent  from  at  least  50  c.c.  of  spring  or  deep 
well  water,  possibly  from  greater  amounts. 

In  upland  surface  waters  the  presence  of  B.  coli  in  40,  10,  or  even 
2  or  1  c.c.  means  contamination,  but  not  necessarily  a  contamination 
which  it  is  essential  to  prevent.  It  may  be  from  contamination 
with  the  excreta  of  animals  grazing  on  the  gathering  areas,  and  is 
by  no  means  necessarily  from  sewage  or  other  material  containing 
specific  organisms  of  infection.  If  B.  coli  are  present  in  numbers 
greater  than,  say,  500  per  litre  (or  even  in  that  amount),  such  a 
water  is  suspicious,  as  it  is  rare  to  get  so  many  B.  coli  in  a  water 
from  the  kind  of  animal  contamination  indicated,  and  further 
investigation  is  desirable.  In  filtered  samples  the  number  of 
B.  coli  is,  as  a  rule,  considerably  reduced. 

In  surface  wells  B.  coli  in  large  numbers  indicate  surface  or  other 
'contamination,  generally  very  undesirable  if  not  actually  dangerous. 

It  must  clearly  be  understood  that  the  presence  of  the  B.  coli 
in  water  is  used  as  an  index  of  pollution,  just  as  the  organic  ammonia 
is  in  a  chemical  analysis.  This  organism  is  not  necessarily  harmful 
in  itself  ;  it  is  what  it  indicates,  viz.  pollution,  probably  with  human 
excremental  matters,  which  may  contain  the  organisms  of  specific 
disease,  e.g.  typhoid,  dysentery,  and  cholera.  As  a  routine,  the 
typhoid  bacillus  is  never  looked  for,  and  the  statement  sometimes 
seen  in  the  report  on  the  bacteriological  examination  of  a  sample 
of  water  that  "  no  typhoid  bacilli  have  been  detected  "  is  of  little 
value.  It  is  on  the  general  results  of  the  examination,  as  detailed  in 
preceding  pages,  that  a  conclusion  is  arrived  at  respecting  the  purity 
or  otherwise  of  a  water. 

Bacillus  Welchii. — This  organism  being  abundantly 
present  in  fseces  and  sewage,  its  presence  in  water  has  been 
suggested  as  an  indication  of  pollution.  Its  spores,  how- 
ever, are  very  resistant,  and  it  might,  therefore,  gain 
access  to  the  water  in  ways  other  than  by  direct  pollution — 
e.g.  in  dust — and  for  this  reason  the  committee  did  not 
recommend  the  search  for  this  organism  as  a  routine 


EXAMINATION  OF  WATER  589 

procedure.  On  the  other  hand,  Thresh  l  lays  a  good  deal 
of  stress  on  it,  and  the  following  are  standards  suggested 
by  him,  based  on  an  examination  for,  and  detection  of, 
B.  coli  and  B.  Welchii  : 

1.  Water  showing  the  absence  of  organisms  capable  of  fermenting 
glucose,  and  of  the  B.  Welchii.     These  we  regard  as  being  free  from 
any  evidence  of  pollution. 

2.  Waters  showing  the  absence  of  organisms  capable  of  fermenting 
glucose,  but  containing  the  B.  Welchii,  or  its  near  ally.     In  the  few 
cases  of  this  kind  which  have  come  under  our  observation  we  have 
inferred  the  absence  of  sewage  pollution,  but  the  possible  presence 
of  water  derived  from  fertile  soil.     This  inference  has  been  verified 
on  more  than  one  occasion. 

3.  Waters  containing  organisms  capable  of  fermenting  glucose, 
but  not  lactose,  but  free  from  the  spores  of  the  B.  Welchii.     These 
are  regarded  as  unpolluted. 

4.  Waters  differing  from  No.  3  only  in  containing  spores  of  the 
B.   Welchii.     These  we  regard  as  free  from  sewage  pollution,  but 
as  probably  containing  soil  washings. 

5.  Waters  containing  lactose  fermenters,  none  of  which  belongs 
to  the  Bacillus  coli  group,  and  free  from  the  spores  of  the  B.  Welchii. 
These  we  do  not  regard  as  being  sewage-polluted,  but  as  containing 
surface  water  or  subsoil  washings. 

6.  Waters  resembling  No.  5,  but  containing  the  spores  of  the 
B.  Welchii.    These  waters  are  usually  from  a  source  requiring  careful 
watching,  manurial  matter  probably  being  used  on  the  collecting 
area. 

7.  Waters  containing  organisms  of  the  colon  group  other  than 
the  B.  coli,  but  no  spores  of  the  B.  Welchii.     These  we  do  not  regard 
as  dangerously  polluted,  but  as  probably  coming  from  a  source 
such  as  that  referred  to  under  No.  6. 

8.  Waters  containing  organisms  of  the  colon  group  other  than 
the  B.  coli,  and  also  spores  of  the  B.  Welchii.     Pollution  indicated, 
but  possibly  from  a  source  not  close  at  hand.     The  necessity  for 
frequent  examination  is  essential,  especially  after  heavy  rains,  as 
such  waters  usually  sooner  or  later  show  more  serious  signs  of 
pollution. 

9.  Waters  containing  the  true  B.  coli,  but  no  spores  of  the  B. 
Welchii.     Such  waters  are  occasionally  met  with.     No  opinion  can 
be  expressed  without  an  intimate  knowledge  of  the  source.     We 

1  Public  Health,  1904. 


590  A  MANUAL  OF  BACTERIOLOGY 

have  had  such  water  from  a  source  absolutely  free  from  the  possi- 
bilities of  contamination,  but  usually  subsequent  examination 
has  revealed  the  presence  of  the  spores  of  the  B.  Welchii.  The 
proximity  of  manured  soil  is  strongly  indicated. 

10.  Waters  containing  the  true  B.  coli  and  spores  of  the  B.  Welchii. 
These  we  regard  as  being  decidedly  contaminated  with  faecal  matter 
of  recent  origin. 

Streptococci. — Streptococci  are  abundant  in  faeces  and 
sewage,  but  are  extremely  rare,  if  ever  present,  in  unpolluted 
natural  waters  ;  hence  the  value  of  their  detection.  Strep- 
tococci as  a  class  are  delicate  organisms,  and  it  was  supposed 
that  their  presence  indicates  recent  pollution.1  Horrocks, 
on  the  other  hand,  believes  that  they  maintain  their 
vitality  longer  even  than  B.  coli,  and  this  is  rather  the 
opinion  at  present.  We  need  further  data  before  we  can 
exactly  estimate  the  value  of  streptococci  as  indicators  of 
pollution.  There  can  be  no  question,  however,  that  the 
detection  of  many  streptococci,  together  with  B.  coli, 
indicates  serious  pollution. 

There  can  be  no  doubt  of  the  value  of  the  bacteriological  examina- 
tion of  water,  but  it  cannot  entirely  supplant  chemical  analysis, 
which  on  account  of  its  rapidity  and  the  valuable  data  it  yields 
will  probably  always  remain  an  integral  part  of  the  examination 
of  potable  waters.  If  the  water  be  pure  and  uncontaminated,  the 
bacteriological  examination  will  occupy  three  days  ;  but  if  con- 
tamination be  present,  though  it  may  be  presumed  in  the  same  time, 
ten  days  or  a  fortnight  may  be  required  to  convert  this  presumption 
into  a  certainty,  owing  to  the  length  of  time  necessary  for  deter- 
mining the  characters  of  the  organisms  present. 


Media  Employed  for  the  Isolation  of  B.  Coli 

(1)  Carbolised  gelatin. — Ordinary  nutrient  gelatin  with  the  addi- 
tion of  0-05  per  cent,  of  phenol.     (Hardly  used  now.) 

(2)  Bile-salt  peptone  water  (MacConkey  and  Hill). — The   com- 
position of  this  medium  is  as  follows  :  Sodium  taurocholate  0-5  grm., 
glucose  or  lactose  1-0  grm.,  peptone  2-0  grm.,  water  100  c.c.     The 

1  Houston,  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1898-99. 


ISOLATION  OF  BACILLUS  COLI  591 

constituents  are  dissolved  by  heating  ;  the  mixture  is  filtered,  and 
after  filtration  sufficient  neutral  litmus  solution  is  added  to  give  a 
distinct  colour.  The  medium  is  then  distributed  into  Durham's 
fermentation-tubes  and  sterilised  by  steaming  for  twenty  minutes 
on  three  successive  days.  The  medium  may  be  put  up  in  various 
sized  tubes,  a  measured  volume  in  each — e.g.  10  c.c.,  20  c.c.,  25  c.c., 
etc.,  according  to  the  quantity  of  water  which  is  to  be  added.  For 
the  larger  quantities  the  medium  may  be  made  double  the  above 
strength.  The  inoculated  tubes  are  incubated  at  42°  C.  for  forty- 
eight  hours.  The  B.  coli  reddens  and  ferments  both  the  glucose 
and  lactose  media,  so  that  gas  collects  in  the  fermentation  tube. 

(3)  Neutral-red  broth  (Hunter,  Makgill,  Savage). — The  dye  known 
as  neutral-red  (Griibler's)  is  reduced  by  the  action  of  the  B.  coli, 
the  colour  changing  to  a  canary  yellow,  accompanied  by  a  green 
fluorescence.     The  B.   enteritidis  (Gartner)  also  reduces  neutral- 
red,  but  the  B.  typhosus  does   not    do  so,  nor   do   streptococci, 
B.  pyocyaneus,  and  Vibrio  cholerce.     Some  anaerobes  also  possess 
a  reducing  action.     Glucose  agar  or  broth  (0-5  per  cent,  of  glucose) 
is  employed,  and  to  every  10  c.c.  of  the  medium  0-1  c.c.  of  a  0-5 
per  cent,  aqueous  solution  of  neutral-red  is  added.     Savage  recom- 
mends the  following  procedure :    10  c.c.  of  the  water  are  added  to 
a  10  c.c.  tube  of  neutral-red  broth  ;    also  to  40  c.c.  of  the  water 
contained  in  a  bottle  or  flask  a  10  c.c.  tube  of  the  broth  of  quadruple 
strength  is  added.     Both  are  incubated  at  37°  C.,  and  examined 
daily  up  to  eight  days.     If  reduction  occurs,  B.  coli  is  almost 
certainly  present  in  the  water  ;    if  reduction  does  not  occur  its 
presence  is  highly  improbable. 

(4)  Glucose  formate  broth  (Pakes). — To  ordinary  meat  infusion 
1  per  cent,  peptone,  0-5  per  cent,  sodium  chloride,  2  per  cent,  glucose, 
and  0-4  per  cent,  sodium  formate  are  added.     When  these  have 
been  dissolved  by  heating,  the  medium  is  neutralised  (indicator, 
litmus),  and  after  neutralisation  2  c.c.  of  normal  caustic  soda  solu- 
tion per  litre  are  added  ;    the  broth  is  then  steamed  for  twenty 
minutes,  filtered,  and  distributed  into  test-tubes,  10  c.c.  in  each, 
which  are  steamed  for  twenty  minutes  on  each  of  three  successive 
days.     These  tubes  are  inoculated  with  the  water,  and  incubated 
anaerobically  at  42°  C.  for  twenty-four  to  seventy-two  hours.     Tubes 
showing  any  growth  at  the  end  of  twenty-four,  forty-eight,  or 
seventy-two  hours  are  removed  and  examined  microscopically  and 
by  plating. 

(5)  Bile-salt  lactose  agar  (MacConkey). — This  medium  is  prepared 
by  adding  to  1000  c.c.  of  tap-water  in  a  flask  2  per  cent,  of  peptone, 
0-5  per  cent,  of  sodium  taurocholate,  and  1-5  per  cent,  of  agar. 


592  A  MANUAL  OF  BACTERIOLOGY 

The  mixture  is  autoclaved  at  105°  to  110°  C.  for  1^  hours,  cleared 
with  a  small  addition  of  white  of  egg,  and  filtered.  To  the  nitrate 
1  per  cent,  of  lactose  is  added.  The  medium  is  then  distributed 
into  test-tubes,  10  c.c.  in  each,  and  sterilised  by  fifteen  minutes' 
steaming  on  three  successive  days.  Plates  are  made  and  incubated 
at  42°  C.  for  forty-eight  hours.  The  colonies  of  organisms  which 
ferment  lactose  with  the  formation  of  acid  are  surrounded  with  a 
cloudiness  or  haze  owing  to  the  precipitation  of  the  taurocholate. 
Neutral-red  or  krystal  violet  may  be  added  (proportions,  see  Nos.  3 
and  6). 

(6)  Conradi-Drigalski  agar.  Mixture  A. — To  1  litre  of  acid 
beef  broth  (p.  54)  add  : 

Witte's  peptone        .          .          .          .          .10  grm. 

Nutrose  10      „ 

Sodium  chloride        .          .          .          .  5      „ 

Steam  for  one  hour,  and  add  25  grm.  of  powdered  agaf.  Steam 
for  three  hours,  bring  to  a  reaction  of  +  10,  and  filter  through 
"  papier  Chardin." 

Mixture  B. — Boil  for  a  few  minutes  100  c.c.  of  Kubel-Tiemann's 
litmus  solution,  add  15  grm.  of  pure  powdered  lactose,  and  boil 
again  for  a  few  minutes. 

Add  B  to  A,  and  to  this  mixture  add  2  c.c.  of  a  hot  10  per  cent, 
solution  of  anhydrous  sodium  carbonate  and  10  c.c.  of  a  0-1  per  cent, 
solution  of  krystal  violet.  The  medium  is  then  tubed,  10  c.c.  being 
placed  in  each  test-tube,  and  sterilised. 

In  using  the  medium  it  should  be  employed  as  surface  plates. 
The  required  number  of  tubes  are  melted  in  a  water-bath,  and 
their  contents  poured  out  into  sterile  Petrie  dishes  and  allowed  to 
set.  These  sterile  plates  are  then  placed  in  the  warm  incubator 
for  an  hour  or  so  with  the  lids  slightly  tilted  at  one  edge,  so  that 
the  surface  of  the  medium  may  dry  somewhat.  The  matter  to 
be  plated  is  sufficiently  diluted,  and  from  a  few  drops  to  0-5  c.c. 
are  run  on  to  the  surface  and  spread  by  means  of  a  glass  rod  bent 
into  a  flattened  hook,  and  sterilised  by  boiling.  On  this  medium 
in  forty-eight  hours  B.  coli  forms  large  red  colonies,  B.  typhosus 
and  B.  dy sentence  small  blue  colonies,  and  streptococci  small  delicate 
red  colonies.  Other  organisms  are  to  a  large  extent  inhibited  from 
developing. 

(7)  8.D.8.  rebipelagar  (Houston). — "  Rebipelagar "  has  been 
much  used  by  Houston  *  for  the  isolation  of  B.  coli.  It  has  the 
following  composition  :  Agar  20  grm.,  taurocholate  of  soda  5  grm., 

1  First  Rep.  on  Research  Work,  Met.  Water  Board,  1908. 


SPECIFIC  ORGANISMS  IN  WATER          593 

lactose  10  grm.,  neutral-red  4  c.c.  of  a  1  per  cent,  solution,  peptone 
20  grm.,  water  1  litre.  The  S.D.S.  rebipelagar  has  the  following 
composition :  Agar  20  grm.,  taurocholate  of  soda  5  grm.,  lactose 
2-5  grm.,  neutral-red  4  c.c.  of  a  1  per  cent,  solution,  peptone  20  grm., 
saccharose  2-5  grin.,  dulcitol  2-5  grm.,  salicin  2-5  grm. 


The  Isolation  of  Specific  Organisms 
from  Water 

The  principal  disease-producing  organisms  conveyed  by  water 
are  the  B.  typhosus,  B.  paratyphosus,  B.  dysenterice,  and  Vibrio 
cholerce. 

THE    ISOLATION     OF    B.     TYPHOSUS,    B.     PARATYPHOSUS,     AND    B. 

DYSENTERIC  FROM  WATER. — There  is  great  difficulty  in  isolating 
the  B.  typhosus  from  water  that  has  been  very  copiously  contami- 
nated with  specifically  polluted  sewage,  there  is,  therefore,  far 
greater  difficulty  when  the  specific  pollution  has  been  small  in 
amount.  The  earlier  records  of  the  isolation  of  the  B.  typhosus 
must  be  accepted  with  much  scepticism,  as  the  methods  of  identi- 
fication were  formerly  incomplete  and  unsatisfactory.  It  is  neces- 
sary to  bear  in  mind  that  usually,  when  drinking-water  has  suffered 
sewage-pollution,  the  amount  of  the  pollution  is  relatively  very 
minute  when  compared  with  the  great  bulk  of  the  water-supply. 
Moreover,  allowing  ten  days  as  the  average  incubation  period  of 
typhoid  fever,  another  week  before  the  disease  comes  under  notice, 
and  another  week  before  the  fact  that  an  epidemic  is  in  progress 
is  recognised,  at  least  a  month  will  have  elapsed  between  the  date 
of  infection  of  the  water-supply  (supposing  this  to  have  occurred 
on  one  occasion  only,  as  may  be  the  case)  and  the  taking  of  the 
samples  for  examination,  a  period  during  which  all  the  typhoid 
bacilli  may  have  died  out.  The  contamination  of  water  may, 
however,  be  of  an  intermittent  nature. 

Numerous  methods  *  have  been  devised  for  the  isolation  of  the 
typhoid  bacillus  from  an  infected  water.  With  rare  exceptions, 
it  is  impossible  to  detect  the  organism  by  direct  plating  ;  it  is  too 
scanty  and  too  mixed  with  other  organisms  to  admit  of  this,  and 
therefore  concentration  of  the  bacterial  content  of  the  water  must 
be  attempted.  The  following  are  some  of  the  methods  which  have 
been  suggested  for  this  purpose  ;  they  serve  equally  well  for  B. 
paratyphosus  and  B.  dysenterice. 

1  See  H.  S.  Willson,  Journal  of  Hygiene,  vol.  v,  1905,  p.  429 ; 
McWeeney,  Brit.  Med.  Journ.,  1909,  vol.  ii,  p.  866. 

38 


594  A  MANUAL  OF  BACTERIOLOGY 

1.  Filtration  through  a  porcelain  filter. — By  passing  one  to  two 
litres  of  the  water  through  a  sterile  Pasteur-Chamberland  filter, 
the  whole  of  the  organisms  present  may  theoretically  be  collected 
in  a  few  c.c.s.      Practically,  however,  a  large  proportion  of  the 
organisms  are  lost  in  the  process :    perhaps  they  get  carried  into 
and  remain  in  the  superficial  layers  of  the  filter-candle,  and  for  this 
reason,  though  sometimes  employed,  this  method  has  been  largely 
given  up. 

2.  Concentration. — W.    J.    Wilson 1    has    devised   the   following 
method :    The  water  is  placed  in  one  or  two  Winchester    quart 
bottles,  and  10  c.c.  of  nutrient  broth  are  added  for  every  litre.     The 
bottles  are  placed  in  a  water- bath  maintained  at  37°-40°  C.,  and 
are  connected  by  rubber  corks  and  tubing  with  a  condenser  (at  a 
lower  level)  through  which  cold  water  continuously  passes,  and 
the  tube  of  the  condenser  is  connected  to  a  large  bottle  (at  a  still 
lower  level).     This  bottle  is  kept  partially  exhausted  by  means  of 
a  filter-pump.     The  water  evaporates  and  is  thus  concentrated,  the 
evaporated  water  being  condensed  and  collected  in  the  exhausted 
bottle.     It  requires  twenty-one  to  twenty-two  hours  to  evaporate 
a  litre  of  water.     The  water  remaining  in  the  bottles,  now  concen- 
trated to  a  few  c.c.s.,  is  then  plated  on  Conradi-Drigalski  or  mala- 
chite-green agar. 

3.  Chemical  precipitation. — These  methods  depend  on  the  forma- 
tion in  the  water  of  a  fine,  inert  precipitate,  which  entangles  and 
carries  down  with  it  a  large  proportion  of  the  bacteria  present.     Thus 
in  the  Vallet-Schiider  2  method,  to  2  litres  of  the  water  are  added 
20  c.c.  of  a  7-75  per  cent,  solution  of  sodium  hyposulphite  and 
20  c.c.  of  a  10  per  cent,  solution  of  lead  nitrate.     The  precipitate 
is  allowed  to  settle  or  is  centrifuged  off,  is  dissolved  in  a  small 
volume  of  a  saturated  solution  of  the  hyposulphite,  from  which 
plates  are  made  in  suitable  media.     Ficker  3  uses  ferrous  sulphate 
after  making  the  water  faintly  alkaline  with  caustic  soda  ;    the 
ferrous   hydrate  formed  carries   down  the  micro-organisms   (this 
must  be  a  risky  procedure,  as  the  typhoid  bacillus  is  very  sensitive 
to  caustic  alkalies).     Iron  oxychloride  may  also  be  used  as  the 
precipitant.     H.    S.    Willson   (loc.   cit.)   employs   alum.     A   stock 
solution  of  alum  is  prepared,  containing  10  grm.  per  100  c.c.,  and 
of  this  sufficient  is  added  to  the  water  to  obtain  0-5  grm.  to  the 
litre.     After  the  precipitate  of  aluminium  hydrate  has  formed, 
the  vessel  is  well  shaken  to  mix  its  contents,  and  the  mixture  is 

1  Brit.  Med.  Journ.,  1907,  vol.  i,  p.  1176. 

2  Zeitschr.f.  Hyg.,  xlii,  No.  2,  p.  317. 

3  Hyg.  Rundschau,  xiv,  No.  1,  1904.  p.  7. 


ISOLATION  OF  BACILLUS  TYPHOSUS        595 

centrifuged  for  fifteen  minutes  at  2000  revolutions  per  minute. 
The  clear,  supernatant  fluid  is  then  syphoned  or  poured  carefully 
off  from  the  precipitate,  and  the  mass  of  precipitate  in  the  conical 
extremity  of  the  tube  stirred  up  with  the  little  fluid  (0-5  to  1  c.c.) 
remaining.  The  suspension  is  then  plated  out  on  Conradi-Drigalski, 
malachite -green  or  brilliant -green,  agar.  This  seems  to  be  a  very 
promising  method. 

4.  Serum  agglutination. — An  anti-typhoid  serum — the  serum  of 
an  animal  which  has  been  inoculated  several  times  with  the  typhoid 
bacillus,    having  the   power  of   agglutinating  typhoid   bacilli — if 
added  to  a  water  would  presumably  agglutinate  any  typhoid  bacilli 
into  masses  which  will  sediment  or  may  be  centrifuged  off.     The 
method  has  been  used  by  Schepilewsky,1  who  adds  10  to  20  c.c.  of 
the  water  to  flasks  containing  50  c.c.  of  nutrient  broth,  to  which 
after  three  or  four  days  incubation  at  37°  C.  an  addition  of  the 
typhoid  serum  is  made,  and  after  standing  for  some  hours  and 
centrifuging,  the  deposit  is  plated  out. 

5.  Method  of  enrichment. — The  principle  of  this  method  is  to 
devise  a  medium  which  will  allow  of  the  multiplication  of    the 
typhoid  bacillus   and  at  the  same  time  prevent,  or  at  least  retard, 
the  growth  of  B.  coli  and  allied  forms.     Almost  all  the  methods 
which    have  been  introduced  for  this  purpose  fail,  inasmuch  as 
though  they  inhibit  the  growth  of  a  great  many  organisms,  they 
do  not  inhibit  the  growth  of  the  B.  coli,  or,  if  they  do,  inhibit  the 
B.  typhosus  to  a  still  greater  degree.     Roth  2  found  that  caffeine 
in  broth  would  retard  B.  coli,  but  allow  B.  typhosus  to  multiply. 
The  method  has  been  further  elaborated  by  Hoffmann  and  Ticker,3 
who  convert  the  water  itself  into  a  nutrient  medium  by  the  addition 
of  1  per  cent,  of  nutrose,  0-5  per  cent,  caffeine,  and  0*001  per  cent, 
of  krystal  violet.     The  mixture  is  incubated  at  37°  C.  for  not  more 
than  twelve  to  thirteen  hours,  at  the  end  of  which  time  the  typhoid 
bacilli  should  have  multiplied  to  such  an  extent  as  to  permit  of 
direct  isolation  by  plating,   the  B.   coli  being  inhibited.     Many 
observers  have  shown,  however,  that  while  caffeine  may  materially 
help,  it  cannot  be  entirely  relied  on  to  eliminate  B.  coli  and  allied 
forms. 

6.  Process  of  Cambier. — Cambier  4  has  devised  a  process  based  on 
the  idea  that  an  actively  motile  organism  will  find  its  way  through 
the  pores  of  a  porcelain  filter  more  quickly  than  feebly  or  non- 

1  Centr.  f.  Bakt.,  Orig.,  xxiii,  No.  5,  1903. 

2  Hyg.  Rundschau,  xiii,  1903,  p.  489. 

3  Ibid,  xiv,  1904,  p.  1. 

4  Rev.  dHyg.,  1902,  p.  64. 


596  A  MANUAL  OF  BACTERIOLOGY 

motile  forms.  His  procedure  is  to  make  use  of  a  special  alkaline 
peptone  medium,  which  is  placed  in  a  glass  jar.  In  this  is  immersed 
a  Pasteur-Chamberland  filter-candle  half  filled  with  the  same 
solution,  to  which  is  added  a  little  of  the  fluid  to  be  examined,  and 
the  whole  is  incubated  at  37°  C.  Sooner  or  later  growth  appears 
in  the  fluid  outside  the  candle,  and  Cambier  states  that  if  typhoid 
bacilli  be  present  they  will  make  their  appearance  before  B.  coli. 
In  hands  other  than  those  of  Cambier,  however,  the  method  has 
not  proved  successful. 

7.  Fuchsin  agar  (Endo). — One  litre  of  3  per  cent,  nutrient  agar 
is  made  alkaline  with  10  c.c.  of  10  per  cent.  NaOH  solution  after 
neutralisation.     Pure   lactose    10    grm.    and    saturated    alcoholic 
fuchsin  solution  5  c.c.  are  added,  and  after  mixing,  25  c.c.  of  fresh 
10  per  cent,  solution  of  sodium  sulphite  are  added.     The  medium 
when  cold  should  be  colourless.     The  medium  is  used  as  surface 
plates,  and  on  it  typhoid  and  paratyphoid  colonies  are  colourless, 
coli  colonies  are  red. 

8.  Malachite-green   media. — Loffler    has   found    that    malachite 
green  (No.  120  Hoechst)  in  the  proportion  of  about  1  in  5000  in 
media  inhibits  the  growth  of  B.  coli  while  still  permitting  the 
growth  of  B.  typhosus.     The  dye  may  be  added  either  to  liquid  or 
to  solid  media.     The  medium  recommended  by  Loffler  l  is  com- 
posed of  3  per  cent,  agar  made  with  meat  infusion,  with  1  per  cent, 
nutrose,  and  containing  in  every  100  c.c.  2-2-5  c.c.  of  a  1  per  cent, 
solution  of  malachite  green.     On  this  medium  the  B.    typhosus 
grows  in  twenty-four  hours  as  delicate,  slightly  crinkled  colonies, 
surrounded  by  a  colourless  zone  (due  to  alkali  formed  by  the  bacilli). 
Thus  it  is  possible  to  detect  one  colony  of  B.  typhosus  among  300  to 
600  colonies  of  other  bacteria.     As  a  medium  for  "  enriching  "- 
i.e.  for  specially  advancing  the  growth  of  the  B.  typhosus — Loffler 
recommends  a  15  per  cent,  gelatin,  prepared  with  beef -juice  and 
peptone,  and  containing  per  100  c.c.  3  c.c.  of  doubly  normal  phos- 
phoric acid  and  2  c.c.  of  2  per  cent,  malachite-green  solution.     With 
the  suspected  matter,  firstly,  one  series  of  malachite -gelatin  plates 
is  prepared  and  incubated  at  25°  C.  for  twenty  to  twenty-four 
hours  ;    secondly,  a  tube  of  malachite  gelatin  is  inoculated  and 
incubated  at  37°  C.  for  twelve  to  twenty-four  hours  ;  from  this  a 
second  tube  is  inoculated  and  incubated  at  37°  C.,  and  then  plated 
out  on  malachite  gelatin  and  incubated  at  25°  C.     The  colonies  of 
B.  typhosus  are  well  marked  after  twenty  to  twenty -four  hours,  as 
large  as  a  pin's  head,  transparent,  highly  refractile,  light  grey  and 
granular.     Their  shape  is  circular  or  oval,  and  they  show  charac- 

1  Deutsch.  med.  Woch.,  1906,  No.  8. 


ISOLATION  OF  BACILLUS  TYPHOSUS        597 

teristic  offshoots  resembling  a  bone-corpuscle  or  the  body  of  an 
acarus.  By  using  this  15  per  cent,  gelatin,  which  can  be  incubated 
at  25°  C.,  there  is  the  double  advantage  of  speedy  growth  and 
formation  of  very  characteristic  colonies. 

Houston  recommends  S.D.S.  rebipelagar  (p.  592)  with  the  addi- 
tion of  malachite -green  to  the  extent  of  1  in  5000  (0-2  grm.  to  the 
litre).  On  this  medium  B.  typhosus  forms  colourless  colonies ; 
most  other  bacteria  do  not  grow,  or  appear  as  blue-black  colonies. 

9.  Werbitzlci's   China  green  agar. — For  this  3  per  cent,  nutrient 
agar  (reaction   +13)  is  used,  and  to  every  100  c.c.  of    the  agar 
1-4-1-5   c.c.  of    a  0-2  per  cent,  aqueous  solution  of   china  green 
(Griibler's)  are  added. 

10.  Brilliant  green  agar. — Conradi  devised  an  agar  containing 
brilliant  green  and  picric  acid,  and  this  has  been  modified  by  Fawcus  * 
as  follows  :  To  900  c.c.  of  tap-water  are  added  sodium  taurocholate, 
5  grm.  ;  powdered  agar,  30  grm.  ;  peptone,  20  grm.  ;  and  sodium 
chloride,  5  grm.     Dissolve  the  constituents  by  steaming  for  three 
hours,  filter  through  wool,  and  bring  to  a  reaction  of  +  15  (by 
means  of  lactic  acid  or  NaOH,  as  the  case  may  be).     In  100  c.c.  of 
distilled  water  dissolve  10  grm.  lactose  and  add  this  to  the  former 
filter,  distribute  in  flasks  (100  c.c.  in  each),  and  sterilise.     At  time 
of  using,  melt  and  add  to  each  100  c.c.,  2  c.c.  of  a  1-1000  aqueous 
solution  of  brilliant  green  and  2  c.c.  of  a  1-100  aqueous  picric  acid 
(extra-pure,  Griibler's).     Typhoid  forms  round,  transparent  refrac- 
tile  colonies  of  a  light  pale  green  colour  by  transmitted  light,  B.  colt 
dark  green  colonies  with  an  opaque  spot  at  the  centre. 

CONCLUSION. — The  writer  would  suggest  for  the  isolation 
of  B.  typhosus  from  water  :  (1)  Concentration  of  the 
organism  by  precipitation  with  alum  (Willson's  method) 
or  iron  oxychloride,  followed  by  plating  of  the  precipitate 
on  Conradi-Drigalski  agar,  or,  better,  on  malachite  green 
agar  (Loffler's  or  Houston's,  No.  8  above),  or  brilliant- 
green  agar  (No.  10  above)  ;  (2)  enrichment  by  Loffler's 
method  and  subsequent  plating.  In  all  cases  the  organism 
isolated  must  be  examined  as  to  its  morphological,  cultural, 
and  biological  characters,  and  should  have  its  agglutination 
and  Pfeiffer  reactions  tested  with  a  high-grade  typhoid 
serum.  Two  organisms  which  are  likely  to  be  mistaken 

1  Journ.  Roy.  Army  Med.  Corps,  February  1906,  p.  147. 


598  A  MANUAL  OF  BACTERIOLOGY 

for  the  B.  typhosus,  unless  all  tests  are  applied  to  them, 
are  the  B.  (fcecalis)  alkaligenes  and  B.  (aquatilis)  sulcatus. 
Both  occur  in  the  dejecta  and  in  polluted  water,  and  are 
very  like  the  B.  typhosus  in  morphology,  motility,  staining, 
and  cultural  reactions,  but  neither  agglutinates  with 
typhoid  serum.  The  B.  alkaligenes  sometimes  produces 
a  brownish  growth  on  potato,  it  renders  litmus  milk 
alkaline  and  produces  alkali,  but  no  gas,  in  glucose,  lactose, 
dulcitol,  mannitol,  saccharose,  and  salicin.  The  B.  sulcatus 
hardly  grows  at  37°  C.  and  is  almost  a  strict  ae'robe,  little 
growth  occurring  in  the  depth  of  a  stab.  Some  varieties 
of  typical  and  of  atypical  B.  coli  agglutinate  with  typhoid 
serum,  so  that  a  positive  agglutination  reaction  does  not 
necessarily  prove  that  an  organism  is  B.  typhosus. 

THE  ISOLATION  or  THE  CHOLERA  BACILLUS  FROM  WATER. — The 
detection  of  Koch's  comma  bacillus  (Vibrio  cholerce)  in  water,  as 
in  the  case  of  the  typhoid  bacillus,  is  a  matter  of  some  difficulty, 
as  this  organism  is  rapidly  overgrown  by  the  ordinary  water  bacteria. 
In  the  examination  of  suspected  water  supplies,  the  best  method 
to  employ  for  the  detection  of  this  organism  is  to  take  advantage 
of  the  fact,  first  noted  by  Dunham,  that  the  cholera  vibrio 
multiplies  with  great  rapidity  in  alkaline  saline  peptone  solution. 
The  suspected  water  is  examined  as  follows :  To  300-500  c.c.  of 
the  water  are  added  1  per  cent,  each  of  pure  peptone  and  of  common 
salt ;  the  mixture  is  made  faintly  alkaline  with  sodium  carbonate, 
distributed  in  a  dozen  small  Erlenmeyer  flasks  having  a  layer  not 
more  than  an  inch  deep  in  each,  the  flasks  are  loosely  capped  with 
caps  of  filter-paper,  and  incubated  at  37°  C.  At  intervals  of  ten, 
fifteen  and  twenty  hours  respectively,  hanging- drop  and  cover-glass 
preparations  are  made  from  the  top  of  the  liquid,  an  which  there 
is  often  a  surface  film,  and  care  must  be  taken  not  to  disturb  this  ; 
these  are  then  examined  microscopically  for  vibrios  and  spirilla. 
At  the  same  time  agar  (3  per  cent.),  or,  better,  blood  alkali  agar 
(p.  446)  plates  are  prepared  and  incubated  at  blood-heat.  Any 
colonies  that  appear  which  resemble  the  cholera  spirillum  are 
examined  microscopically  ;  if  the  organisms  are  comma-shaped, 
they  are  at  once  subcultured  into  peptone  water  and  other  media. 
The  original  peptone  water  cultures  are  tested  for  the  indole  reaction 
with  pure  hydrochloric  acid,  withdrawing  some  of  the  contents  of 


STERILISATION  OF  WATER  599 

the  flasks  with  a  sterile  pipette.  Any  likely  vibrios  isolated 
must  have  its  cultural  and  biological  reactions  investigated  and  be 
tested  for  the  agglutination  and  Pfeiffer  reactions  with  a  high-grade 
cholera  serum. 

On  the  survival  of  the  typhoid  and  cholera  organisms  in  water, 
see  pp.  360  and  437  respectively. 

Ice  and  ice-creams  may  be  examined  by  methods  similar  to  those 
used  for  water,  the  material  being  first  melted  at  a  low  temperature. 
Some  of  the  fluid  should  also  be  centrifuged  and  the  deposit 
examined  microscopically  for  gross  contamination. 

The  infection  in  typhoid  fever  and  cholera,  and  perhaps 
also  in  bacillary  dysentery,  is  perhaps  more  frequently 
water-borne  than  conveyed  in  any  other  way.  It  might 
be  supposed  that  the  acid  gastric  juice  would  prevent  this, 
and  it  may  do  so  in  many  instances.  Experiments  by 
Macfadyen l  showed  that,  whereas  in  fasting  animals, 
to  which  suspensions  in  water  of  the  cholera  vibrio  were 
administered,  living  vibrios  pass  into  the  intestine,  when 
the  vehicle  is  milk  none  could  be  detected  in  the  intestines. 
The  inference  is  that  when  there  is  no  food  there  is  no 
gastric  juice  secreted  and  the  organisms  are  able  to  pass 
into  the  intestine,  but  when  food  is  present  the  gastric 
juice  is  secreted  and  the  organisms  are  destroyed. 

STERILISATION  OF  WATER. — This  may  be  done  on  the  small  scale 
by  heat,  by  the  use  of  germicidal  agents,  or  by  filtration  through  a 
filter  (see  p.  601).  Heat  may  be  applied  by  simple  boiling,  or  by 
the  use  of  apparatus  in  which  the  water  is  heated  to  65°-90°  C., 
and  the  outgoing  hot  water  is  cooled  by  the  ingoing  cold  water, 
which  itself  is  thus  warmed,  thereby  effecting  economy  in  fuel 
(Griffiths'  and  other  sterilisers).  The  chemical  germicides  that 
have  been  employed  are  (1)  sodium  bisulphate,  15  grains  to  the 
pint  ;  (2)  Potassium  permanganate,  sufficient  to  tinge  the  water 
deeply  for  at  least  half  an  hour  ;  (3)  chlorine  gas  or  iodine  tablets,2 
in  both  cases  the  taste  of  the  agent  being  destroyed  by  the  addition 
of  sodium  sulphite  ;  (4)  copper  and  copper  sulphate.  Sufficient 
metal  is  dissolved  from  bright  copper  in  twenty-four  hours  to  destroy 

1  Journ.  of  Anat.  and  PhysioL,  vol.  xxi. 

2  Nesfield,  Journ.  Prev.  Med.,  vol.  xiii,  1905,  p.  623. 


600  A  MANUAL  OF  BACTERIOLOGY 

typhoid  and  cholera.  Copper  sulphate  1  in  100,000  or  less  is 
similarly  germicidal,  and  in  still  smaller  quantities  (1  in  1,000,000) 
destroys  algae,  and  has  been  used  for  the  purification  of  reservoirs 
overgrown  with  algae.  On  the  large  (also  small)  scale,  chlorine 
derived  from  hypochlorites  is  one  of  the  simplest  and  most  efficient 
agents.  Moor  and  Hewlett  *  showed  that  0-25  part  of  chlorine 
(equivalent  to  about  0-75  part  of  good  chloride  of  lime)  per  million 
parts  of  chalk  water  is  sufficient  to  kill  B.  coli  in  half  an  hour. 
The  taste  disappears  quickly  in  bright  sunlight  and  on  standing, 
or  may  be  removed  by  an  addition  of  sodium  sulphite.  If  the 
water  is  organically  polluted,  more  chlorine  must  be  used. 

Ozone  produced  by  high-tension  electric  discharge  is  also  employed 
on  the  large  scale  for  the  sterilisation  of  water-supplies,  e.g.  at 
Chartres  (see  also  p.  637). 

EXAMINATION  OF  SHELL-FISH. — Shell-fish  may  come  from  sewage- 
polluted  layings  (see  p.  362).  The  following  method  may  be 
employed  for  their  examination  (after  Houston) : 

The  outside  of  the  shells  are  cleansed  by  thorough  scrubbing  and 
rinsing  in  tap-water,  and  a  final  rinse  in  sterile  water.  The  fish 
after  cleansing  are  laid  on  a  sterile  towel.  The  operator  then 
cleanses  his  hands  and  opens  the  shells  aseptically  with  a  sterile 
oyster-knife,  care  being  taken  to  avoid  loss  of  their  contained  liquor. 
The  liquor  as  each  fish  is  opened  is  poured  into  a  sterile  litre  cylinder, 
and  the  fish  is  cut  up  with  sterile  scissors  and  added  to  the  liquor 
in  the  cylinder.  Ten  fish  should  be  treated,  the  volume  of  fish  -f- 
liquor  noted,  and  sterile  water  is  then  added  to  make  up  to  1  litre  ; 
100  c.c.  liquid  therefore  corresponds  to  one  fish.  In  addition, 
four  dilutions  of  the  liquid  are  prepared — 1  in  10,  1  in  100,  1  in 
1000,  and  1  in  10,000.  With  the  liquid  and  dilutions  gelatin  and 
agar  plate  cultivations  are  prepared  for  the  enumerations  of  the 
organisms  present.  Cultures  are  also  made  in  litmus  lactose  bile- 
salt  peptone  water  and  in  milk  for  the  enumeration  and  isolation 
of  B.  coli  and  B.  Welchii  respectively,  taking  100  c.c.,  10  c.c.,  and 
1  c.c.  of  the  liquid,  and  1  c.c.  of  each  of  the  four  dilutions  ;  in  this 
way  the  contents  of  the  fish,  ranging  from  one  fish  to  TTnrl___  of 
a  fish,  are  examined.  The  process  and  principles  involved  corre- 
spond to  those  described  for  water.  Houston  has  suggested  for 
oysters  as  a  lenient  standard  less  than  1000,  and  as  a  stringent 
standard  less  than  100,  B.  coli  per  oyster.  Even  ten  B.  coli  per 
fish  should  be  viewed  with  suspicion,  for  Hewlett  and  others  have 
shown  that  oysters  from  pure  layings  contain  no  B.  coli. 

Watercress,  etc.,  may  be  examined  in  a  similar  manner,  100  grm. 
1  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1909-10,  p.  559. 


FILTERS  601 

being  weighed  out  and  transferred  bit  by  bit  with  sterilised  forceps 
and  scissors  to  a  flask  containing  900  c.c.  of  sterile  water.  The 
flask  is  shaken  vigorously,  and  the  washings  examined  in  a  manner 
similar  to  that  employed  for  shell-fish. 

FILTERS. — Reference  has  already  been  made  to  the 
removal  of  organisms  in  water  by  sand  nitration.  With 
regard  to  niters  for  domestic  use,  few  of  those  in  the  market 
are  capable  of  doing  more  than  removing  particles  of 
suspended  matter,  while  they  allow  from  5  to  50  per  cent., 
or  even  more,  of  the  bacteria  present  in  the  water  to  be 
filtered,  to  pass  through.  Such  filters  are,  of  course, 
useless  for  the  prevention  of  disease — in  fact,  rather  favour 
it,  by  engendering  a  false  sense  of  security ;  and  when  in 
use  for  some  time  without  cleaning,  the  water  after  filtra- 
tion may  be  worse,  bacteriologically  and  chemically,  than 
before  filtration. 

Woodhead  and  Wood  x  found  that  the  only  filters  which 
were  capable  of  completely  removing  organisms  were  the 
Pasteur-Chamberland,  Berkefeld,  and  Porcelaine  d'Amiant. 
The  Berkefeld,  while  more  rapid  in  action  than  the  other 
two,  after  being  in  use  for  a  few  days  may  allow  some 
organisms  to  appear  in  the  filtrate.  This,  perhaps,  is  due 
rather  to  a  growth  of  organisms  through  the  pores  of  the 
filter-candle  than  to  a  direct  passage.  Lunt  2  found  that 
while  the  ordinary  water  bacteria,  such  as  the  B.  fluorescens 
liquefaciens,  appeared  in  the  filtrate  from  a  Berkefeld  filter 
within  a  few  days  of  the  infection  of  the  sample,  the  typhoid 
bacillus  and  the  comma  bacillus  similarly  introduced  had 
not  passed  through  the  filter  four  or  five  weeks  after 
infection. 

Horrocks,3  however,  does  not  confirm  this,  and  has 
found  that  when  sterile  water  is  inoculated  with  typhoid 

1  Brit.  Med.  Journ.,  1894,  vol.  ii,  p.  1053  et  seq. 

2  Trans.  Brit.  Inst.  of  Prev.  Med.,  vol.  i,  1897. 

3  Brit.  Med.  Journ.,  1901,  vol.  i,  p.  1471. 


602  A  MANUAL  OF  BACTERIOLOGY 

bacilli  and  run  daily  through  a  Berkefeld  filter,  the  bacilli 
appear  in  the  nitrate  in  one  or  two  weeks,  whereas  this  is 
not  the  case  with  the  Pasteur-Chamberland.  The  writer 
has  made  some  similar  experiments,  which  partially,  but 
not  entirely,  support  Horrocks's  conclusions.  Much 
evidently  depends  upon  the  chemical  composition  of  the 
water. 

Messrs.  Doulton  have  constructed  a  porcelain  filter  which 
seems  to  be  perfectly  efficient,  like  the  Pasteur-Chamber- 
land. All  porcelain  niters  should  be  cleaned  weekly  by 
well  scrubbing  with  a  nail-brush  and  boiling  in  water 
containing  some  sodium  carbonate. 

The  Bacteriological  Examination  of  Water- 
Filters 

The  large  majority  of  water-filters  at  present  in  use  are  incapable 
of  preventing  organisms  from  being  washed  through  into  the 
filtrate.  In  order  to  ascertain  whether  this  is  the  case  with  any 
particular  filter,  it  should  be  sterilised  in  the  steam  steriliser,  and 
water  containing  organisms  of  known  species  (B.  prodigiosus,  B. 
violaceus,  and  M.  agilis  are  very  suitable)  should  be  passed  through 
it  for  twenty-four  hours.  This  water  and  the  filter  should  during 
this  period  of  the  examination  be  maintained,  if  conveniently 
possible,  at  a  temperature  below  5°  C.  This  will  almost  invariably 
prevent  any  growth  or  multiplication  of  the  organisms.  Samples 
should  be  taken  immediately  after  the  filtration  has  begun,  and  at 
intervals  during  the  day,  and  again  at  the  end  of  twenty -four  hours. 
If  they  are  all  sterile,  the  filter  is  capable  of  preventing  organisms 
from  being  directly  washed  through.  In  the  case  of  filters  of  very 
great  density  or  depth  of  filtering  medium,  it  may  be  necessary  to 
prolong  the  period  of  examination  beyond  the  first  day  ;  but  most 
ordinary  filters  which  permit  organisms  to  be  washed  through  do 
so  within  the  first  few  hours. 


Protozoa  and  Algae  in  Water 

The    examination  of  water  for  the  minute  forms  of  life  other 
than  bacteria,  and  their  enumeration,  can  be  carried  out  by  the 


BACTERIOLOGY  OF  AIR  603 

Sedgwick-Rafter  method.1  A  6-inch  glass  funnel  is  plugged  at  the 
bottom  of  the  stem  with  a  perforated  rubber  cork,  over  the  upper 
end  of  which  a  disc  of  fine  silk  bolting  cloth,  cut  by  a  wad-cutter, 
is  laid.  Sharp,  clean,  dry  quartz  sand  is  then  poured  into  the 
stem  of  the  funnel  to  the  depth  of  half  an  inch  above  the  plug. 
The  sand  should  be  of  such  a  size  that  the  grains  will  pass  through 
a  sieve  of  60  meshes  to  the  inch,  but  not  through  one  of  120  meshes. 
The  sand  is  washed  into  place  and  well  moistened  with  a  little 
distilled  water  free  from  organisms. 

The  water  to  be  examined  is  thoroughly  shaken  and  500  c.c. 
are  poured  into  the  funnel ;  it  runs  through  the  sand,  which  detains 
any  organisms  it  may  contain.  After  the  water  has  all  passed 
through,  the  rubber  plug  is  carefully  removed  and  the  sand  washed 
down  into  a  test-tube  with  5  c.c.  of  distilled  water.  The  contents 
of  the  test-tube  are  agitated  and  the  tube  is  allowed  to  rest  until 
the  sand  has  deposited.  Immediately  this  is  the  case  the  super- 
natant fluid  is  decanted  into  a  second  test-tube,  carrying  with  it 
the  organisms.  One  cubic  centimetre  of  this  is  withdrawn  by  a 
pipette  from  midway  between  the  top  and  bottom  and  transferred 
to  the  counting  plate.  This  consists  of  an  ordinary  glass  slide  on 
which  a  rectangular  brass  cell  (20  x  50  mm.)  is  cemented,  so 
enclosing  exactly  1000  square  mm.  The  brass  cell  is  1  mm.  thick, 
so  that  the  cell  contains  exactly  1  c.c.  The  preparation  is  covered 
with  a  cover-glass  and  examined  with  a  low  power.2 


The  Bacteriology  of  Air 

Just  as  in  water,  the  bacteria  in  the  air  vary  considerably 
at  different  times  and  seasons,  under  different  conditions, 
and  in  various  localities.  The  species  met  with  are  mostly 
saprophytes,  consisting  largely  of  chromogenic  forms.  A 
number  of  moulds  occur  (as  spores),  and,  in  fact,  ordinarily 
are  in  large  excess,  together  with  yeasts  and  torula?. 

It  is  not  easy  for  micro-organisms  to  become  diffused 
through  the  atmosphere  ;  they  are  incapable  of  a  volun- 
tary rising,  and  cannot  be  torn  from  a  fluid  or  moist  solid 

1  Calkin,    Twenty-third  Ann.   Rep.   State  Board   of    Health,   Massa- 
chusetts, 1891. 

2  On  the  microscopy  of  water,   see  Whipple,  Microscopy  of  Drinking 
Water, 


604  A  MANUAL  OF  BACTERIOLOGY 

medium  by  a  strong  current  of  air.  The  medium  on 
which  they  are  growing  must  dry  up  completely  and  crumble 
into  fine  dust  before  they  can  be  distributed  through  the 
agency  of  air-currents  (but  see  p.  365). 

The  number  of  organisms  in  the  air  varies  with  the 
season,  with  rain,  with  altitude,  with  movement,  etc.  At 
Montsouris,  Miquel  found  in  one  cubic  metre  of  air  49 
organisms  in  winter,  85  in  spring,  105  in  summer,  and 
142  in  autumn.  After  heavy  rain  the  air  is  largely  freed 
from  organisms.  Frankland  found  at  Norwich  Cathedral 
at  an  altitude  of  300  feet  7  organisms  in  two  gallons,  while 
on  the  ground  18  were  found ;  at  the  Golden  Gallery  at 
St.  Paul's  two  gallons  of  air  contained  11  organisms  ;  in 
St.  Paul's  churchyard  the  number  was  70.  On  high 
mountains  organisms  are  nearly  absent  from  the  air,  and 
the  same  is  the  case  at  sea  at  a  distance  from  land  exceeding 
about  100  miles.  Organisms  are  much  fewer  in  the  air 
of  the  country  than  in  that  of  towns.  At  the  entrance-hall, 
Natural  History  Museum,  South  Kensington,  Frankland 
found  in  the  morning  30  organisms  ;  in  the  afternoon, 
when  many  visitors  were  present,  the  number  had  risen 
to  292,  showing  the  influence  of  movement.  By  keeping  a 
volume  of  air  absolutely  still,  enclosed  in  a  box  the  walls 
of  which  were  smeared  with  glycerin,  Tyndall  was  able 
to  free  it  completely  from  particles  and  organisms.  The 
writer  found  from  43  to  150  organisms  per  10  litres  of  air 
in  some  of  the  principal  streets  of  London  during  the 
daytime. 

Gordon,1  by  exposing  dishes  of  neutral-red  broth  to  the 
air,  or  by  aspirating  air  through  neutral-red  broth  (p.  591) 
has  been  able  to  detect  the  presence  of  the  S.  salivarius, 
M.  epidermidis,  and  scurf  micrococcus  (p.  230)  in  air 
subjected  to  human  contamination.  By  these  tests  and 
by  the  use  of  B.  prodigiosus  as  an  indicator  he  concludes 
1  Reps.  Med.  Off.  Loc.  Gov.  Board  for  1902-1904. 


EXAMINATION  OF  AIR  605 

that  particles  of  saliva  are  disseminated  as  far  as  40  feet 
in  the  act  of  loud  speaking,  indicating  the  possibility  of 
the  wide  distribution  of  such  pathogenic  organisms  as 
the  tubercle,  plague,  and  influenza  bacilli  and  the  pneumo- 
coccus  by  speaking,  and  still  more  so  by  coughing. 

The  number  of  dust  particles  in  the  air  may  be  enormous. 
In  London  Macfadyen  and  Lunt  observed  as  extremes  from 
20,000  to  nearly  600,000  per  c.c.  The  ratio  of  micro- 
organisms to  dust  particles  is  therefore  a  very  small  one. 


Bacteriological  Examination  of  Air 

A  number  of  methods  have  been  devised  for  the  estimation  of 
the  number  of  micro-organisms  in  the  air,  of  which  the  following 
are  the  principal  ones  : 

(1)  Plate  method. — Melted  sterile  nutrient  gelatin  is  poured  into 
a  sterilised  Petri  dish,  and  allowed  to  set.     The  plate  is  then  exposed 
to  the  air,  by  removing  the  lid,  for  a  given  time — one,  five,  ten, 
or  fifteen  minutes,  etc. — the  lid  is  replaced,  and  the  plate  incubated 
at  22°  C.  for  some  days.     The  number  of  colonies  of  moulds,  bacteria, 
yeasts,   etc.,  is  counted,   and,   having  estimated  the  area  of  the 
gelatin  plate,1  the  result  is  expressed  as  the  number  of  organisms 
falling  per  square  foot  per  minute.     The  results  obtained  by  this 
method  are  roughly  comparative,  but  no  estimate  can  be  formed 
from  it  of  the  number  of  organisms  contained  in  a  given  volume  of 
the  air. 

(2)  Hesse's  method. — This  is  a  quantitative  method  for  estimating 
the  number  of  organisms  contained  in  a  given  volume  of  air.     The 
apparatus  consists  of  a  glass  tube  30  in.  long  by  1^  to  2  in.  in  diameter. 
One  end  of  this  tube  is  plugged  with  a  rubber  cork  through  which 
a  glass  tube  passes,  the  other  end  is  covered  with  a  piece  of  sheet 
rubber  perforated  with  a  hole  £  to  |  in.  in  diameter  ;   over  this  is 
placed  another  sheet  of  rubber,  unperforated.     The  small  tube 
being  plugged  with  cotton-wool,  the  whole  is  sterilised  for  an  hour 
in  the  steam  steriliser.     Just  before  use  40  to  50  c.c.  of  melted 
sterile  nutrient  gelatin  are  poured  into  the  tube,  and  its  walls 
coated  with  the  medium.     The  tube  is  then  strapped  horizontally 
on  to  a  tripod  stand,  and  the  small  tube  connected  by  means  of  a 

1  The  area  of  a  circular  dish  is  calculated  by  multiplying  the  square 
of  the  diameter  by  0'785. 


606  A  MANUAL  OF  BACTERIOLOGY 

piece  of  rubber  tubing  to  an  aspirator  consisting  of  two  flasks 
arranged  so  as  to  form  a  reversible  syphon.  A  litre  of  water  is 
poured  into  the  flask  connected  with  the  tube,  and  the  outer  sheet 
of  rubber  having  been  removed  from  the  end  of  the  tube,  the  water 
is  syphoned  over  to  the  second  flask,  placed  at  a  lower  level,  and 
an  equal  volume  of  air  is  thus  aspirated  through  the  tube.  The 
second  flask  is  then  connected  with  the  tube,  and  the  position 
of  the  flasks  being  reversed  the  water  is  again  syphoned  over  and 
a  second  litre  of  air  passes  through  the  tube,  and  this  process  is 
repeated  until  5,  10,  15,  or  20  litres  of  air  have  been  drawn  through 
the  tube.  The  rate  of  flow  is  controlled  by  a  screw-clamp  on  the 
rubber  connecting-tube  ;  it  should  not  exceed  half  a  litre  per  minute. 
With  this  rate  of  flow  all  the  organisms  are  deposited  on  the  gelatin- 


A  B  c 

FIG.  67. — Frankland's  tube  for  air  analysis. 

coated  tube.  The  aspiration  being  completed  the  rubber  tube  is 
disconnected  and  the  sheet  of  rubber  replaced  over  the  end  of 
the  tube,  which  is  then  incubated,  and  the  colonies  are  counted 
when  they  have  developed. 

(3)  Petri's  method. — Petri  aspirates  the  air  through  a  glass  tube 
containing  sterilised  sand,  kept  in  place  by  fine  wire -gauze  wads. 
When  the  sample  has  been  taken  the  sand  is  distributed  in  Petri 
dishes,  and  melted  sterile  gelatin  is  poured  over  it  and  allowed  to 
solidify,   plate   cultures   being  thus   prepared.     The   objection  to 
this  method  is  the  presence  of  the  opaque  particles  of  sand  in  the 
culture  medium. 

(4)  Frankland's  method. — The  air  to  be  examined  is  aspirated 
through  a  tube  5  in.  in  length  and  £  in.  in  diameter  (Fig.  67).     One 
end  of  the  tube  is  open,  the  other  (c)  is  plugged  with  cotton-wool. 
At  a  distance  of  1  in.  from  the  open  end  the  tube  is  slightly  con- 
stricted to  support  a  plug  of  glass  wool  (A).     At  a  distance  of  2^  in. 
from  this  plug  the  tube  is  again  constricted  to  support  a  second 
plug  (B),  consisting  of  glass-wool  and  finely  powdered  cane-sugar, 
supported  in  front  and  behind  by  plugs  of  glass-wool.     Several 
such  tubes  having  been  prepared,  they  are  placed  in  a  tin  box  and 
sterilised  at  130°  C.  for  three  hours,  and  can  then  be  easily  trans- 
ported without  risk  of  contamination.     When  required  for  use, 
a  tube  is  quickly  removed  from  the  box,  being  handled  by  the 


EXAMINATION  OF  AIR 


607 


plugged  end,  which  is  connected  by  stout  rubber  tubing  to  aspi- 
rating flasks  such  as  are  used  in  Hesse's  apparatus.  The  tube  is 
clamped  horizontally  to  a  retort  stand,  and  by  attaching  the  second 
flask  to  a  small  hand  exhaust-pump,  the  water  can  be  syphoned 
over  from  the  first  flask,  a  corresponding  volume  of  air  passing 
through  the  tube.  When  the  desired  volume  of  air  has  been 
aspirated  through  the  tube,  it  is  disconnected  j-«rt^ 

and  placed  in  another  sterile  tin  box.  As  many 
tubes  as  desired  can  be  employed  to  control 
one  another  or  to  examine  the  air  in  different 
localities  and  under  different  conditions.  All 
the  samples  having  been  taken,  the  tubes  are 
manipulated  on  returning  to  the  laboratory. 
The  tubes,  as  before,  being  handled  by  the 
ends  only,  a  file-mark  is  made  across  the  centre 
of  each  tube,  which  is  then  broken  in  half  and 
the  plugs  of  glass-wool  and  sugar  are  shaken, 
or  pushed  by  means  of  a  sterile  wire,  into  a 
sterile  flask  of  about  250  c.c.  capacity.  Into 
this  10  or  15  c.c.  of  liquefied  sterile  nutrient 
gelatin  are  then  introduced  ;  the  sugar  dis- 
solves, the  glass-wool  becomes  disintegrated, 
and  a  roll-culture  is  made  on  the  walls  of  the 
flask,  which  is  incubated  at  22°  C.,  and  the 
colonies  are  counted  when  they  have  deve- 
loped. 

(5)  Sedgwick  and  Tucker's  method. — One  of 
the  best  and  most  convenient  methods  for  the 
bacteriological  examination  of  air.  A  glass 

tube   of  special  form  is   employed  (Fig.  68)  ;  pIG>  68. Sedgwick 

this  consists  of  an  expanded  portion  (A)  about  and  Tucker's  tube 
15  cm.  long  and  4-5  cm.  in  diameter  ;  one  end  of  for  air  analysis, 
this  is  contracted  so  as  to  form  a  neck  2-5  cm.  in 
diameter  and  in  length  ;  to  the  other  end  is  fused  a  glass  tube  (B  c) 
15  cm.  long  and  0-5  cm.  in  diameter.  The  neck  of  the  tube  is  plugged 
with  cotton-wool,  and  two  cotton-wool — or,  better,  glass-wool — plugs 
are  inserted  in  the  narrow  tube,  one  at  its  open  end,  the  other  (c) 
about  6  to  8  cm.  from  the  wide  part.  The  whole  is  then  sterilised. 
When  cool,  the  narrow  part  of  the  tube,  from  its  origin  at  the  wide 
part  down  to  the  first  plug  (c),  is  filled  with  powdered  cane-sugar 
(No.  50,  B.P.  gauge)  which  has  been  carefully  dried  and  sterilised 
at  120°-130°  C.  The  tube  is  again  sterilised  at  120°-130°  for  two 
or  three  hours,  the  greatest  care  being  taken  not  to  melt  the  sugar. 


608  A  MANUAL  OF  BACTERIOLOGY 

After  sterilisation  the  tube  is  ready  for  use.  The  wool  plug  is 
removed  from  the  mouth  and  a  measured  volume  of  air  is  aspirated 
through  the  layer  of  powdered  sugar  by  means  of  a  small  hand 
air-pump,  the  volume  of  air  being  measured  by  the  displacement 
of  water  in  a  flask.  Having  taken  the  sample  (5  to  20  litres),  the 
wool  plug  is  replaced  in  the  neck.  The  powdered  sugar  is  then 
shaken  down  into  the  wide  part  of  the  tube  (A),  and  15  c.c.  of  melted 
sterile  nutrient  gelatin  are  poured  in.  The  powdered  sugar  readily 
dissolves  in  the  melted  gelatin,  and  when  solution  is  complete  a 
roll-culture  is  made  in  the  tube,  just  as  in  Esmarch's  method  (p.  83). 
The  tube  is  then  placed  in  an  incubator  at  20°  C.,  and  the  colonies 
are  allowed  to  develop. 

In  both  Frankland's  and  Sedgwick  and  Tucker's  methods  the 
sugar,  after  powdering  and  sifting  and  before  introducing  into  the 
tubes,  should  be  thoroughly  dried  by  keeping  in  the  warm  incubator 
for  several  days  ith  occasional  stirring.  Unless  this  be  done,  the 
sugar  is  apt  to  cake  and  discolour  during  sterilisation. 


Soil 

The  upper  layers  of  soil  contain  large  numbers  of  organisms, 
chiefly  bacilli.  The  species  are  very  varied  ;  among  pathogenic 
ones  may  be  named  the  bacillus  of  tetanus  and  of  malignant  oedema. 
The  B.  mycoides  is  very  abundant,  and  the  varieties  of  Proteus, 
the  hay  and  potato  bacilli,  are  common,  while  the  nitrifying  forms 
are  of  course  present,  but  do  not  develop  on  ordinary  media. 

Below  five  or  six  feet  aerobic  organisms  become  scanty,  but  the 
anaerobic  and  thermophilic  ones  are  still  met  with.  The  number 
of  organisms  present  in  soil  is  variable,  from  200,000  to  45,000,000 
in  ordinary  earth,  while  in  dirty  and  busy  streets  there  may  be  as 
many  as  1,000,000,000  per  grm.  According  to  Houston,  unculti- 
vated sandy  soil  averages  100,000,  garden  soil  1,500,000,  and  sewage 
polluted  115,000,000  per  grm. 

Houston  *  found  that  in  virgin  soils  the  B.  coli,  B.  Welchii,  and 
streptococci  are  practically  absent,  but  that  in  soils  polluted  with 
animal  excrement  by  manuring  or  otherwise  the  spores  of  B.  Welchii 
are  present  in  great  abundance,  also  B.  coli  and  streptococci  if  the 
pollution  be  of  recent  date. 

The  length  of  time  pathogenic  bacteria  retain  their  vitality  in 
buried  corpses  has  been  the  subject  of  experiment  by  Losener,2 

1  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1889-1900. 

2  Centr.f.  Bakt.  (VQ  Abt.),  xx,  1896,  p.  454. 


EXAMINATION  OF  SOIL  609 

who  injected  cultures  into  the  bodies  of  pigs,  which  were  then 
wrapped  in  linen,  placed  in  wooden  coffins,  and  buried.  The 
conclusions  he  arrived  at  were  that,  provided  the  soil  has  good 
filtering  properties,  there  is  practically  no  chance  of  the  dissemina- 
tion of  a  virus. 

Klein,1  experimenting  with  the  bacilli  of  diphtheria,  cholera, 
plague,  typhoid  fever,  etc.,  also  found  that  the  vitality  and  infective 
power  of  these  organisms  passed  away  in  a  comparatively  short 
time,  in  most  cases  within  a  month. 

On  the  survival  of  the  typhoid  and  cholera  organisms  in  soil 
see  also  pp.  363  and  437  respectively. 


Examination  of  Soil 

The  bacteria  in  the  soil  may  be  examined  by  adding  traces  of  the 
soil  to  sterile  nutrient  broth,  thoroughly  crushing  and  soaking  it, 
and  then  making  plate  or  roll  cultures,  aerobic  and  anaerobic. 

To  make  anything  like  an  accurate  quantitative  examination  is 
almost  impossible.  Weighed  amounts  of  the  soil,  after  thorough 
pulverisation  in  an  agate  mortar,  may  be  introduced  into  sterile 
test-tubes  and  thoroughly  exhausted  by  repeated  washing  with 
sterile  water  or  broth,  plate  cultivations  being  made  with  the 


Various  forms  of  boring  apparatus  have  been  devised  for  with- 
drawing soil  from  different  depths. 


Sewage  2 

Sewage  is  exceptionally  rich  in  organisms,  but  the  numbers  present 
are  variable.  Jordan  in  Massachusetts  found  an  average  of  708,000 
per  cubic  centimetre.  Laws  and  Andrewes  found  from  905,000 
to  11,216,000,  the  latter  being  the  highest  number  obtained.  The 
number  of  organisms  naturally  varies  at  different  seasons  and 
with  the  amount  of  dilution.  The  organisms  present  are  very 
varied,  but  moulds,  yeasts,  and  sarcinse  only  occasionally  occur. 
A  few  micrococci  are  met  with  and  streptococci  are  present  in 
considerable  numbers,  at  least  1000  per  c.c.,  but  bacilli,  especially 
liquefying  forms,  largely  predominate.  The  commonest  species 

1  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1898-99,  p.  344. 

2  See   various   Reports   to  the  London   County  Council    by  Clowes, 
Houston,  Laws  and  Andrewes ;  Klein,  Houston,  Reps.  Med.  Off.  Loc. 
Gov.  Board  for  1897-1904  ;  Rep.  of  the  Sewage  Commission. 

39 


610  A  MANUAL  OF  BACTERIOLOGY 

are  the  B.  fluorescens  liquefaciens  and  varieties,  several  varieties  of 
Proteus,  the  B.  filamentosus,  varieties  of  the  B.  mesentericus,  B. 
mycoides,  B.  subtilis,  B.  cloacce,  and  the  colon  bacillus.  The  latter 
numbers  from  20,000  to  2,000,000  per  c.c.,  and  the  other  bacilli 
mentioned  number  200,000  to  2,500,000  per  c.c.  Many  anaerobic 
sporing  bacilli  are  also  found,  especially  the  B.  Welchii,  the  spores 
of  which  number  from  30  to  2000  per  c.c.,  averaging  500-600. 
Foreign  bacteria  introduced  into  sewage  are  probably  soon  sup- 
pressed by  the  predominant  species  of  the  sewage. 

The  air  of  well- ventilated  sewers  differs  but  little  from  that  of 
the  external  air,  and  the  organisms  in  it  contrast  with  those  of 
sewage  by  the  abundance  of  moulds.  Specific  organisms  may, 
however,  gain  access  to  it  (p.  365). 

The  powerful  liquefying  and  solvent  actions  of  the  bacteria 
present  in  sewage  have  suggested  a  means  of  dealing  with  sewage 
so  as  to  make  use  of  these  properties,  and  many  bacterial  systems 
of  sewage  disposal  have  been  devised.  The  principle  most  widely 
adopted  is  to  run  the  sewage  into  large  covered  reservoirs  (septic 
tanks),  where  it  remains  at  rest  for  twenty-four  to  forty-eight  hours. 
Here  it  is  under  practically  anaerobic  conditions,  and  anaerobic 
bacteria  exert  their  action  on  the  solids,  partly  dissolving  them, 
partly  disintegrating  them,  with  the  formation  of  a  sludge  which 
has  to  be  cleared  out  from  time  to  time.  From  the  septic  tanks 
the  sewage  passes  on  to  beds  composed  of  broken  brick,  coke,  or 
some  similar  material,  through  which  it  slowly  percolates,  and 
here  it  is  subjected  to  the  action  of  aerobic  organisms,  which  com- 
plete the  decomposition  to  such  an  extent  that  the  effluent  does 
not  affect  fish  life  nor  putrefy,  so  that  it  may  be  run  into  a  stream 
without  causing  a  nuisance.  Four  sets  of  these  aerobic  bacterial 
beds  are  usually  provided,  each  set  being  worked  in  turn  for  six 
hours  and  resting  for  eighteen  hours  during  the  twenty-four  hours. 
The  effluent  from  such  bacterial  beds  may  contain  as  many  bacteria 
as,  or  more  than,  the  sewage  itself.  Pathogenic  organisms  may 
be  present  in  it,  for  Houston  found  that  the  B.  pyocyaneus  added 
to  the  beds  soon  appeared  in  the  effluent. 

On  the  survival  of  the  typhoid  and  cholera  organisms  in  sewage 
see  pp.  363  and  437  respectively. 


Examination  of  Sewage  and  Sewage  Effluents 

To  ensure  a  fair  average  sample,  the  sewage  or  effluent  should 
be  collected  in  small  portions  at  intervals.     The  portions  are  mixed, 


EXAMINATION  OF  SEWAGE 


611 


strained  through  muslin,  and  dilutions  of  1  in  10,  1  in  100,  1  in  1000, 
and  1  in  10,000  made  with  sterile  tap-water.  These  are  then 
examined  according  to  the  following  scheme : 


Tests. 

Procedure. 

Amount  of  sewage  in  c.c. 

1.  Total  number  of 

Gelatin  and  agar  plate 

0-001,  0-0001,  0-00001 

bacteria 

cultivations 

2.  Number  of  spores 

Gelatin  plate  cultures 

1-0,  0-1,  0-01 

of  aerobes 

with    material    pre- 

viously heated  to  80° 

C.  for  ten  minutes. 

3.  Number  of  spores 

Agar     plate     cultures 

1-0,  0-1,  0-01 

of  anaerobes 

with    material   pre- 

viously heated  to  80° 

C.  for  ten  minutes 

and  incubated  anae- 

robically 

4.  Number  of  organ- 

Surface gelatin  plates 

0-001,  0-0001,  0-00001 

isms    liquefying 

gelatin 

5.  Spores  of  B.  Wel- 

Milk    cultures    heated 

0-1,  0-01,  0-001 

chii    (enter  itidis 

to    80°    C.    for   ten 

sporogenes) 

minutes    and    incu- 

bated anaerobically 

6.  Number  of  B.  coli 

Surface-plates  of  Con- 

radi  -  Drigalski,    or 

0-001,  0-0001,  0-00001 

bile-salt  media,  etc., 

as      described      for 

water  (p.  591) 

7.  Number  of  strep- 

Surface-pl&tes of  Con- 

0-01,  0-001,  0-0001 

tococci 

radi  -  Drigalski  me- 

dium (p.  592) 

EFFLUENTS  ONLY. 

8.  Incubate  some  of  the  effluent  in  beakers  at  22°  C.  and  37°  C.  for 

some  days.  A  good  effluent  should  yield  little  or  no  unpleasant 
odour  (an  unpleasant  odour  indicates  the  presence  of  decom- 
posable organic  matter,  and  such  an  effluent  might  give  rise  to 
a  nuisance). 

9.  Place  a  gold-fish  or  two  in  a  bowl  of  the  effluent.     The  fish  will 

live  in,  and  be  unaffected  by,  a  satisfactory  effluent.  (This  may 
be  done  only  by  a  licensee  under  the  Vivisection  Act.) 


612  A  MANUAL  OF  BACTERIOLOGY 

Milk1 

Milk  is  an  admirable  nutrient  soil  for  the  development 
and  multiplication  of  micro-organisms,  and,  though  sterile 
in  the  udder,2  as  delivered  to  the  consumer  may  contain 
an  appalling  number  of  bacteria.  In  milk  as  ordinarily 
supplied  there  are  from  one  to  five  million  bacteria  per  c.c., 
and  it  frequently  contains  ten  to  fifteen  millions,  with  an 
average  of  about  three  to  four  millions.  Hewlett  and 
Barton  found  an  average  bacterial  content  of  about 
1,500,000  in  London  milk  as  delivered  at  the  railway  termini 
(the  range  was  from  a  minimum  of  20,000  to  a  maximum 
of  8,390,000),  but  this  does  not  represent  the  condition  of 
the  milk  as  delivered  to  the  consumer,  for  the  bacteria 
present  rapidly  multiply  in  warm  weather.  Eyre  3  in 
the  middle  of  summer  found  the  following  rate  of  multi- 
plication : 

Microbes  per  c.c. 

Initial  content  .  .  .  56,000 

After  12  hours  .  .  .  526,000 

After  24  hours  .  .  .  20,366,000 

After  30  hours  .  .  .  clotted 

A  similar  specimen  in  the  middle  of  winter  gave  the 
following  results  : 

Microbes  per  c.c. 

Initial  content  .  .  .  20,000 

After  12  hours  .  .  .  24,000 

After  24  hours  .  .  .  43,000 

After  30  hours  .  .  .  280,000 

1  See  Houston,  Rep.  to   the  London  County  Council,  No.  933,  1905 ; 
MacConkey,  Journ.  of  Hygiene,  vol.  v,   1905,  p.  333  ;    Hewlett  and 
Barton,  ibid.  vol.  vii,  1907,  p.  22  ;    Savage,  Rep.  Med.  Off.    Loc.  Gov. 
Board  for  1909-10,  p.  474,  and  Milk  and  the  Public  Health  (Macmillan, 
1912)  ;  Swithenbank  and  Newman,  Bacteriology  of  Milk. 

2  The  "  fore  "  milk  may  contain  organisms  which  have  lodged  in  the 
milk-ducts,  and  it  is  extremely  difficult  to  obtain  completely  sterile  milk. 

3  Journal  of  State  Medicine,  vol.  xii,  1904,  p.  728. 


BACTERIAL  CONTENT  OF  MILK  613 

In  New  York,  Park  estimated  the  average  bacterial 
content  of  milk  as  supplied  to  the  consumer  at  1,000,000 
per  c.c.  in  winter  and  5,000,000  per  c.c.  during  the  hot 
months.  Eyre  (loc.  cit.)  states  that,  as  the  result  of  his 
observations,  the  numbers  are  in  London  about  3,000,000 
to  5,000,000  in  December,  January,  and  February,  and 
20,000,000  to  30,000,000  in  June  to  September,  smaller 
numbers  than  these  always  being  associated  with  the 
presence  of  boric  acid  or  formaldehyde.  Even  in  so-called 
sterilised  milks  bacteria  are  rarely  completely  absent. 

Cream  is  even  richer  in  bacteria  than  milk,  and  averages 
about  8,000,000,  and  may  contain  as  many  as  30,000,000 
organisms  per  c.c.  Although  all  the  ordinary  species  may 
be  met  with,  milk  has  a  bacterial  flora  largely  its  own, 
comprising  many  forms  producing  lactic  and  butyric  acid 
fermentations.  Organisms  also  occur  having  more  or 
less  specific  effects,  and  giving  rise  to  bitter  milk,  viscid 
milk,  etc.  The  lactic  ferments  are  mostly  non-sporing, 
the  butyric  chiefly  sporing,  species.  The  commonest  of 
the  lactic  ferments  are  Streptococcus  lacticus  (non-gas- 
forming)  and  B.  acidi  lactici  (gas- forming),  which  has 
some  similarity  to  the  colon  bacillus  (see  Table,  p.  381). 
Another  common  lactic  organism  is  the  Oldium  lactis,  a 
mycelial  form,  the  colonies  of  which  appear  as  little  fluffy 
tufts.  In  addition  to  the  organisms  named,  pathogenic 
species  may  be  met  with — viz.  the  tubercle,  diphtheria, 
typhoid,  paratyphoid,  Gartner,  dysentery,  and  comma 
bacilli,  the  M .  melitensis,  M.  pyogenes,  and  the  Streptococcus 
pyogenes  (lactic-acid-forming  streptococci  are  also  common). 
The  B.  coli  and  B.  Welchii  are  generally  present  in  milk, 
and  the  B.  lactis  aerogenes  is  sometimes  found  (p.  389). 
Scarlatina  (see  "  Scarlatina  ")  and  foot-and-mouth  disease 
may  likewise  be  conveyed  by  milk,  and  the  diarrhoea  of 
infants  is  largely  due  to  the  use  of  milk  swarming  with 
microbes,  some  of  which  in  themselves  may  be  harmful, 


614  A  MANUAL  OF  BACTERIOLOGY 

and  which  also  by  the  products  they  form  tend  to  set  up 
gastro-enteritis.     The  percentage  of  samples  infected  with 
tubercle  bacilli  varies  much  :    Barton  and  Hewlett  found 
only  one  out  of  26  samples  taken  at  London  railway 
termini.     The   supply   of   the   large   dairy   firms   is   also 
comparatively  free   from   tuberculous   infection,   as   con- 
siderable precautions  are  taken  to   exclude  tuberculous 
animals.     For   the   quarter   ending  March   31,    1911,    of 
760  samples  examined  for  the  London  County  Council, 
106,  or  13-9  per  cent.,  were  found  to  be  tuberculous,  and 
since  1907  of  5698  samples,  640,  or  11-2  per  cent.,  proved 
tuberculous  (see  also  p.  321).     A  poisonous  body,  tyro- 
toxicon  (p.  38)  has  been  isolated  from  milk  and  milk 
products.     Sources   of   contamination   and   infection   are 
derived  from  the  insanitary  conditions  of  many  farms  and 
dairies  and  the  dirty  methods  of  those  handling  the  milk. 
In  order  to  render  milk  wholesome  for  infants  and  free 
from  infective  organisms  under  the  present  conditions  of 
supply,  two  methods  may  be  adopted — sterilisation  and 
pasteurisation.     To  ensure  sterilisation  it  is  necessary  to 
heat  the  milk  to  boiling-point  for  six  hours,  or  to  expose 
it  for  a  shorter  period  to  steam  under  pressure.      Such 
treatment, .  however,  markedly  alters  the  flavour  of  the 
milk,  and  is  said  to  diminish  its  nutritive  value.     If  the 
milk  be  heated  to  a  temperature  not  exceeding  70°  C., 
the  flavour  and  nutritive  qualities  are  far  less  altered,  while 
the  pathogenic  species  are  all  destroyed.     This  method  is 
termed  "  pasteurisation,"  and  consists  in  heating  the  milk 
to  about  60°-68°  C.  for  twenty  to  thirty  minutes.     Pas- 
teurisation destroys  92-99  per  cent,  of  the  total  organisms 
present.     The  objections  to  pasteurised  milk  are  that  the 
natural  enzymes  present  in  fresh  milk  are  destroyed  and 
such  heated  milk  is  stated  to  induce  scurvy  rickets,1  the 

1  Dr.  Lane-Claypon  denies  this,  and  considers  that  the  enzymes  in  milk 
are  derived  from  the  bacteria  in  it  (Rep.  to  theLoc.  Gov.  Board,  1913). 


BACTERIOLOGY  OF  MILK 


615 


lactic-acid-forming  organisms  are  killed,  and  if  the  treated 
milk  be  kept,  the  residuum  of  resistant  putrefactive,  etc., 
bacteria  multiply  enormously,  without  obvious  change  in 
the  milk  and  "  returned  "  milk  can  be  utilised  again  and 
again.  Pasteurised  milk  should  be  rapidly  cooled  and  be 
consumed  within  twenty-four  hours  of  treatment.  Behring 
has  advocated  the  addition  of  formaldehyde  to  all  milk 
used  for  the  feeding  of  children.  Another  method  for 
sterilising  milk  is  the  Budde  process,1  in  which  the  milk, 
after  the  addition  of  hydrogen  peroxide,  is  heated  for 
three  hours  to  52°-53°  C.  All  non-sporing  organisms  are 
destroyed,  and  the  added  hydrogen  peroxide  is  decomposed 
into  H90  and  0. 

All  milk  should  be  distributed  in  closed  bottles,  and 
pasteurised  milk  should  be  consumed  within  thirty-six 
hours  of  treatment. 

The  thermal  death -point  of  pathogenic  organisms  in  milk  is 
as  follows  :  2 


Organism. 

Temperature. 

Period  of  Exposure. 

B.  tuberculosis 

60°  C. 

20  min. 

B.  typliosus 

60°  C. 

2  min. 

B.  cliphtherice  . 

60°  C. 

1  min. 

Spir.  cholerce    . 

60°  C. 

1  min. 

B.  dysenteries  . 

60°  C. 

10  min. 

M.fnelitensis  . 

60°  C. 

20  min. 

The  thermal  death-point  of  tubercle  bacillus,  especially  in  milk 
has  been  the  subject  of  some  controversy  (see  also  p.  309).  De  Man 
found  that  an  exposure  of  fifteen  minutes  at  65°  C.  was  necessary 
l£>  destroy  the  infective  properties  of  tuberculous  milk.  Bang,  of 
Copenhagen,  considers  that  pasteurisation  cannot  always  be  relied 
upon,  and  recommends  that  milk  should  be  heated  to  85°  C.  The 
writer  found  that  the  vitality  of  the  ordinary  non-virulent  laboratory 
cultures  was  destroyed  by  a  temperature  of  60°  C.  acting  for  ten 

1  Hewlett,  Lancet,  1906,  vol.  i,  January  27. 

2  Rosenau,  Hygienic  Lab.,  Washington,  Bull.  42,  1908. 


616  A  MANUAL  OF  BACTERIOLOGY 

minutes,  and  that  the  infective  properties  of  tuberculous  sputum, 
tested  on  guinea-pigs,  were  destroyed  by  a  temperature  of  65°  C. 
acting  for  fifteen  minutes  in  five  out  of  six  instances.  Woodhead  s 
experiments  (First  Royal  Commission  on  Tuberculosis)  gave  irregular 
results  which  seem  to  be  explained  by  Theobald  Smith's  careful 
work.1  This  showed  that  tuberculous  milk  was  rendered  non- 
infective  by  heating  to  60°  C.  for  ten  to  fifteen  minutes,  provided 
there  was  no  formation  of  a  surface  scum  ;  the  latter  seems  to  protect 
the  bacilli.  Russell  and  Hastings  2  confirmed  Smith's  experiments, 
and  assert  that  it  is  sufficient  to  heat  milk  to  60°  C.  (140°  F.)  in 
a  closed  receptacle  for  a  period  of  not  less  than  twenty  minutes  in 
order  to  destroy  the  tubercle  bacillus.  The  surface  scum  forms 
on  milk  only  when  it  is  heated  in  contact  with  air  ;  all  pasteurisers, 
therefore,  should  be  closed  vessels.  The  writer  has  devised  a 
simple  form  of  domestic  pasteuriser,  which  is  made  by  Messrs. 
Allen  and  Hanbury. 

The  occurrence  of  so-called  leucocytes  and  pus-cells  in 
milk  must  be  considered.  A  certain  number  of  cells 
resembling  polymorphonuclear  leucocytes  are  always 
present  in  milk,  more  numerous  during  the  first  week  of 
lactation  and  then  accompanied  by  colostrum  corpuscles. 
An  excess  of  these  cells  may  indicate  some  local  inflamma- 
tory affection  of  the  udder,  or,  if  streptococci  and  blood 
are  present  in  addition,  suppuration,  but  not  necessarily, 
for  Russell  and  Hoffman,  and  Revis  have  shown  that  a 
very  large  cell  count  (500,000-1,000,000,  or  even  10,000,000, 
per  c.c.)  may  often  be  obtained  from  quite  healthy  cows. 
The  nature  of  these  cells  has  been  the  subject  of  an  extended 
investigation  by  Hewlett,  Villar,  and  Revis.3  Their  con- 
clusion is  that  the  majority  of  these  cells  are  not  leucocytes, 
but  are  germinal  cells  of  the  secreting  epithelium  of  the 
udder.  Blood  may  also  be  present  transitorily  in  health 
(Revis).  The  presence  of  squamous  epithelial  cells  indi- 
cates desquamation  from  the  teat  or  udder  or  from  the 
hand  of  the  milker — i.e.  want  of  cleanliness. 

1  Journ.  Exper.  Med.,  vol.  iv,  1899,  p.  217. 

2  17 ih  Ann.  Rep.  Wisconsin  Agricult.  Exp.  Station. 

3  Journ.  of  Hygiene,  vols.  ix,  x,  xi,  and  xiii. 


SOUR  MILK  617 

There  is  no  doubt  that  micro-organisms  are  far  more 
abundant  in  milk  as  supplied  to  the  consumer  than  should 
be.  This  arises  from  the  ignorance  and  carelessness  of 
those  charged  with  the  duty  of  providing  and  distributing 
this  important  article  of  diet.  The  udder  and  teats  of 
the  cow  and  the  hands  of  the  milker  (who  should  wear  a 
special  dress)  should  be  wiped  before  milking,  and  all 
vessels  should  be  clean  and  steamed  or  scalded  before  use. 
The  milk  should  be  cooled  at  once,  some  more  efficiently 
closed  vessel  than  the  present  form  of  milk  churn  adopted, 
and  the  milk  not  stored,  but  forwarded  without  delay 
by  the  railway  companies  in  special  refrigerator  vans. 
Distribution  in  bottles  would  be  a  great  improvement. 

The  following  might  be  suggested  as  a  bacteriological 
standard  for  milk  :  *  (a)  Number  of  organisms  not  to 
exceed  1,000,000  per  c.c.  ;  (b)  absence  of  excess  of  leu- 
cocytes or  of  pus- cells  ;  (c)  B.  coli,  B.  Welchii,  and  strep- 
tococci should  not  be  present  in  1  c.c.  or  less;  (d)  the 
sediment  after  centrifuging  should  be  less  than  100 
parts  per  million ;  (e)  the  milk  as  delivered  should  not 
have  a  temperature  above  10°  C. ;  (/)  absence  of  pathogenic 
organisms. 

Sour  milk. — Sour  milk  is  used  as  an  article  of  diet  in  many  parts 
of  the  world,  e.g.  Bulgaria.  In  these  sour  milks  a  particular  micro- 
organism or  a  variety  of  it,  the  B.  bulgaricus  or  "  bacillus  of  Massol," 
is  generally  present  in  association  with  lactic  streptococci.  It  is 
a  large,  pleomorphic,  Gram-positive  bacillus,  non-motile,  non- 
sporing,  growing  best  at  about  40°  C.,  but  only  in  milk  or  in  culture 
media  made  with  milk  or  whey.  It  has  been  much  employed  for 
the  preparation  of  a  soured  milk  which  is  of  considerable  service 
in  the  treatment  of  certain  disorders.2 

1  See  "  Rep.  of  a  Committee  on  Milk  Supply,"  Philad.  Med.  Journ., 
October  1900,  p.  758  ;    Park,    Journ.  of  Hygiene,  vol.  i,  1901,  p.  391  ; 
Houston,  loc.  cit. 

2  See  Hewlett  and  others,  Brit.  Med.  Journ.,  1910,  vol.  ii  (Bibliog.). 


618  A  MANUAL  OF  BACTERIOLOGY 


Examination  of  Milk 

Number  of  organisms  per  c.c. — This  is  carried  out  by  diluting  the 
milk  to  1  in  1000 — 1  in  1,000,000  with  sterile  water,  or  preferably 
nutrient  broth,  as  a  better  mixture  is  obtained.  Plates  are  then 
made  either  in  gelatin  or  in  distilled  water  agar  (1|  grm.  powdered 
agar,  distilled  water  1  litre,  Eastes),  or  preferably  in  both  media. 

B.  coli,  B.  Welchii,  and  streptococci. — These  are  searched  for 
quantitatively  by  the  methods  detailed  for  "  Water  "  (pp.  576-586). 
Amounts  of  milk  in  decreasing  decimal  order  from  100  c.c.  to 
0-000001  c.c.  should  be  examined.  The  B.  coli  must  be  differentiated 
from  B.  lactis  aerogenes  and  B.  acidi  lactici  (see  pp.  389,  381). 

Pathogenic  organisms. — The  detection  of  these,  with  the  exception 
of  the  tubercle  bacillus,  is  difficult  and  uncertain.  In  all  cases 
the  milk  should  be  centrifuged  and  the  deposit  examined. 

1.  For  the  detection  of  the  tubercle  bacillus  *  staining  methods 
are  almost  useless  (except  in  cases  of  advanced  tuberculosis  of  the 
udder  or  when  the  milk  of  a  single  cow  is  examined)  and  inoculation 
must  be  performed.  At  least  250  c.c.  of  the  milk  should  be  centri- 
fuged at  2000  to  2500  revolutions  per  minute  for  an  hour.  As 
many  organisms  become  entangled  in  the  cream,  it  is  advisable  to 
stop  the  machine  after  half  an  hour,  stir  in  the  cream,  and  again 
centrifuge.  The  fluid  is  poured  or  pipetted  off  carefully,  so  as 
not  to  disturb  the  sediment,  leaving  about  3  c.c.  in  the  tube.  The 
sediment  and  the  remaining  fluid  are  then  well  mixed  and  about 
1  c.c.  is  inoculated  subcutaneously  and  intraperitoneally  into 
two  guinea-pigs  respectively  (see  also  p.  329).  For  staining,  a 
process  of  solution  of  the  milk  may  be  employed,  20  c.c.  of  the  milk 
being  mixed  with  1  c.c.  of  a  50  per  cent,  potash  solution,  and  heated 
in  a  water-bath  until  the  solution  turns  brownish  ;  20  c.c.  of  acetic 
acid  are  then  added.  The  mixture  is  shaken,  heated  in  a  water- 
bath  for  three  minutes,  and  centrifuged  for  ten  minutes.  The 
fluid  is  poured  off,  30  c.c.  of  hot  water  are  added  to  the  sediment, 
and  the  mixture  is  again  centrifuged.  Films  are  then  prepared 
from  the  sediment,  and  stained  for  the  tubercle  bacillus  (see  also 
p.  325),  the  films  being  always  treated  with  alcohol  as  well  as  with 
acid. 

Non-pathogenic  acid-fast  bacilli  occur  in  milk  (p.  340). 
2.  The  diphtheria  bacillus  is  searched  for  by  making  serum 
cultures  from,   and  inoculating  guinea-pigs  with,   the  sediment. 

1  See  Delepine,  Rep.  Med.  Off.  Loc.  Gov.  Board  for  1908-09,  p.  134. 


EXAMINATION  OF  MILK  619 

If  a  diphtheroid  organism  is  detected  it  must  be  isolated  and 
examined  by  culture  tests  and  animal  inoculation. 

In  milk  and  cheese  a  bacillus  is  frequently  met  with  closely  resembling 
the  diphtheria  bacillus  in  its  morphological  and  cultural  characters , 
it  is,  however,  quite  non-pathogenic.1 

3.  The  typhoid,  paratyphoid,  Gartner,  and  dysentery  bacilli 
and  cholera  vibrio  may  be  searched  for  by  the  methods  given  for 
"  Water." 

(4)  The  M.   pyogenes  and  the  Streptococcus  pyogenes  may  be 
searched  for  by  means  of  plate  cultures  on  glycerin  agar. 

(5)  Examination  of  sediment. — Houston   and   Savage   (loc.    cit.) 
have  devised  methods  for  the  quantitative  estimation  of  the  sedi- 
ment by  centrifuging  in  special  graduated  tubes.     For  the  micro- 
scopical examination   of    the    sediment    the    milk   is    centrifuged 
for  twenty  minutes  at  1500  revolutions  per  minute,  and  the  upper 
fluid  is  pipetted  or  syphoned  off.     Some  of  the  sediment  should  be 
examined  with  the  f  in.  and  £  in.  objectives  for  the  presence  of 
"  dirt,"  e.g.  hairs,  straw,  etc.     Three  smear  preparations  are  then 
made,  each  with  four  drops  of  the  sediment,  which  are  spread  evenly 
over  three-fourths  of  the  slide.     The  slides  are  air-dried,  and  may 
be  treated  with  a  mixture  of  absolute  alcohol  and  ether  for  ten 
minutes.     One  slide  is  stained  with  Loffler's   blue,   another  by 
Gram's  method  for  streptococci,  and  a  third  by  the  tubercle  method. 
The  Loffler's  blue  specimen  gives  a  general  idea  of  the  number  of 
bacteria  present,  and  of  the  presence  of  cells. 

From  what  has  been  said  above  (p.  616),  considerable  caution 
must  be  exercised  in  stating  the  presence  of  pus-cells.  Streptococci 
present  are  not  necessarily  pathogenic,  as  non-pathogenic  lactic- 
acid-forming  streptococci  are  common.  For  counting  the  number 
of  cells  present,  Revis  2  employs  a  centrifuge  tube  of  10  c.c.  capacity, 
the  lower  third  of  which  is  contracted  to  0-8  cm.  in  diameter,  and 
contains  1  c.c.  The  procedure  is  as  follows : 

In  the  tube  are  placed  5  c.c.  of  the  well-mixed  milk,  diluted  to 
the  10  c.c.  mark  with  0-8  per  cent,  salt  solution.  After  inserting 
a  rubber  stopper  the  contents  are  well  mixed.  The  tube  is  then 
centrifuged  at  about  2000  revolutions  per  minute  for  two  minutes, 
the  cream  is  broken  up  by  violently  shaking  the  upper  part  of  the 
tube,  and  the  rotation  continued  for  four  minutes  longer.  A  glass 
rod,  fitting  roughly  the  narrow  neck  of  the  tube,  is  inserted,  and 
the  major  part  of  the  milk  poured  off,  and  the  upper  part  of  the 
tube  well  rinsed  with  water  to  remove  cream,  etc.  ;  the  contents 

1  See  Scientific  Bull.  No.  2,  Health  Dept.,  City  of  New  York,  1895,  p.  10. 

2  Journ.  of  Hygiene,  vol.  x,  1910,  p.  58. 


620  A  MANUAL  OF  BACTERIOLOGY 

of  the  narrow  end  down  to  within  \  in.  of  the  deposit  are  sucked  out 
with  a  fine  glass  pipette,  the  upper  part  of  the  tube  is  wiped  clean 
and  the  tube  is  then  filled  to  the  10  c.c.  mark  with  salt  solution. 
The  tube,  having  been  violently  shaken  till  all  the  deposit  is  dis- 
tributed through  the  liquid,  is  then  rotated  for  four  minutes,  and 
the  liquid  down  to  within  |  in.  of  the  deposit  again  removed.  In 
the  case  of  small  deposits,  two  to  three  drops  of  saturated  aqueous 
solution  of  methylene-blue  are  added,  and  the  deposit  is  stirred 
up  by  blowing  through  a  fine  glass  capillary  pipette  (which  is 
afterwards  used  for  filling  the  counting  chamber).  After  fifteen 
minutes,  water  is  added  to  the  1  c.c.  mark,  and  counting  done  in 
the  usual  way  with  a  Thoma-Zeiss  blood  counter.  Counting  should 
not  be  restricted  to  the  ruled  spaces,  but  the  field  should  be  so  ar- 
ranged that  a  definite  number  of  squares  is  included,  and  fields  are 
counted  all  over  the  chamber.  At  least  two  different  preparations 
should  be  made  of  the  same  deposit  for  counting. 

FOOD  PoisoNiNG.1 — Apart  from  the  presence  of  the  ordinary 
poisons,  food  may  be  poisonous  on  eating — (a)  naturally,  e.g.  certain 
fish,  (6)  from  the  results  of  the  activity  of  micro-organisms  with 
the  formation  of  toxic  products,  the  ordinary  "  ptomine  poisoning  " 
(see  p.  38),  in  which  case  the  poison  is  pre-formed  and  is  ingested, 
(c)  from  infection  with  certain  organisms,  particularly  B.  enteritidis, 
which  generally  induce  gastro-enteritis.  In  the  last  named,  symp- 
toms do  not  usually  ensue  until  a  lapse  of  twelve  to  forty-eight 
hours  after  the  consumption  of  the  food.  Mayer  and  Mandel 
describe  an  outbreak  following  the  consumption  of  broiled  fish, 
in  which  B.  proteus  was  isolated  from  the  stools  and  was  agglutinated 
by  the  patients'  serum. 

Meat  is  not  likely  to  convey  any  infective  disease  with  the  excep- 
tion of  tuberculosis  and  anthrax.  It  may  be  examined  by  cultures 
and  plate  cultivations,  and  by  inoculation  and  feeding  experiments. 
Tinned  meats,  etc.,  frequently  contain  sporing  organisms  of  the 
B.  subtilis  and  mesentericus  groups.  They  may  be  examined  by 
aerobic  and  anaerobic  cultures,  and  by  feeding  mice.  Poisonous 
ptomines  are  occasionally  present.  The  B.  enteritidis  occurs  in 
meat,  and  causes  a  form  of  poisoning  (see  p.  371  ).2  In  certain 
intoxications  due  to  bad  meat,  known  as  "  botulism,"  Van  Ermengen 
isolated  the  B.  botulinus  (see  p.  427). 

Bread. — Troitzki  states  that  new  bread  contains  no  micro- 
organisms, but  Waldo  and  Walsh  found  that  such  organisms  as 
the  comma  bacillus  are  not  destroyed  by  passing  through  the  ordeal 

1  See  Savage,  Hep.  to  the  LOG.  Gov.  Board,  No.  77,  1913. 

2  See  Savage,  Eep.  Med.  Off.  Loc.  Gov.  Board  for  1909-10,  p.  446. 


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622  A  MANUAL  OF  BACTERIOLOGY 

of  the  baker's  oven.  Cut  bread  forms  a  good  nidus  for  the  develop- 
ment of  pathogenic  organisms. 

The  Bacillus  prodigiosus  may  grow  upon  various  food-stuffs,  and 
give  rise  to  suspicion  of  foul  play.  L.  Parkes  x  describes  cases  of 
diarrhoea  which  he  suggests  were  caused  by  this  organism. 

Butter  contains  from  two  to  forty-seven  millions  of  micro- 
organisms per  gramme.  Tubercle  bacilli  have  been  found  in  butter, 
and  the  comma  bacillus  artificially  introduced  survives  for  over  a 
month.  "  Acid-fast  "  non-pathogenic  forms  also  occur  (p.  340). 

For  the  isolation  of  the  tubercle  bacillus  from  butter  and  cheese 
the  only  certain  method  is  by  inoculation.  Butter  may  be  melted 
and  allowed  to  stand  in  the  incubator  at  37°  C.  for  some  days,  and 
the  sediment  inoculated.  As  this  involves  the  multiplication  of 
septic  organisms,  it  is  preferable  to  centrifuge  the  melted  butter, 
keeping  it  melted  during  the  process,  and  to  inoculate  the  sediment 
immediately. 

Clothing,  etc. — Attempts  have  been  made  to  examine  clothing, 
bedding,  flock,  etc.,  by  bacteriological  methods  for  filth  contamina- 
tion, but  without  much  success. 

1  Brit.  Med.  Journ.,  1905,  vol.  ii,  1330. 


CHAPTER  XXII 

HEAT  — STEAM  DISINFECTION  —  CHEMICAL  DISINFECT- 
ANTS— THEORY  OF  DISINFECTION— METHODS  OF 
DETERMINING  DISINFECTANT  POWER 

Disinfection  x 

NATURAL  agencies  restrain  the  multiplication  of  disease 
organisms,  but  enough  survive  to  determine  the  persistence 
of  infective  diseases,  and  to  call  for  measures  by  which 
communities  attempt  to  cope  with  them.  These  measures 
are  broadly  isolation,  prophylactic  inoculation,  general 
improvement  in  sanitation  and  nutrition,  and  disinfection. 
In  the  present  chapter  the  methods  by  which  the  fourth 
means  of  protection  may  be  applied  are  considered.  Dis- 
infection implies  the  removal  or  the  destruction  of  infective 
properties,  but,  for  practical  purposes,  should  be  understood 
to  mean  the  killing  of  the  infective  organisms  to  which 
those  properties  are  due.  For  this  purpose,  the  two 
agencies  ordinarily  used  are  heat  and  chemical  action, 
though,  in  addition,  other  methods  can  occasionally  be 
employed  for  destroying  or  excluding  micro-organisms. 
Such  are  light,  desiccation,  and  filtration. 

HEAT. — Fire  is  the  simplest  and  most  efficient  agent 
for  destroying  infective  matter.  Burning  should  always 
be  employed  where  possible,  as  for  rags,  old  clothing  or 
bedding,  native  huts,  etc. 

For  surfaces  which  would  not  be  unduly  injured,  such 
as  stables,  pens,  yards,  etc.,  a  torch-fire  generated  by  means 

1  See  Hewlett,  "  Milroy  Lectures,"  Lancet,  1909,  vol.  i. 
623 


624  A  MANUAL  OF  BACTERIOLOGY 

of  the  cyclone  burner  described  by  Forbush  and  Fernald 
has  been  favourably  reported  on  by  Stiles.  The  apparatus 
consists  of  a  portable  tank,  from  which  paraffin  gas  oil 
is  driven  by  a  pump  through  a  hose  (such  as  is  used  for  the 
delivery  of  oil)  to  which  is  attached  a  pole,  consisting  of 
an  iron  pipe  12  ft.  long,  which  is  protected  by  a  covering 
of  wood,  and  to  the  end  of  which  is  attached  a  cyclone 
nozzle.  The  fine  spray  from  the  nozzle  is  ignited,  and  the 
resulting  fierce  flame  passed  over  the  surfaces  to  be  dis- 
infected. The  thorough  wetting  with  water  of  all  such 
surfaces  would  practically  abolish  danger  from  fire,  and 
by  proper  adjustment  of  the  power  of  the  flame,  and 
experience  on  the  part  of  the  operator,  the  method  is  an 
efficient  one. 

Dry  heat  may  also  be  used,  and  forms  the  basis  of  some 
disinfectors  (Ransome's),  but  is  not  nearly  such  an  efficient 
means  as  moist  heat.  The  objections  to  dry  heat  are, 
that  to  ensure  the  destruction  of  bacteria  and  spores  the 
temperature  must  be  high  and  the  heating  prolonged. 
Koch  and  Wolfhiigel  found  that  two  hours  at  150°  C.  did 
not  always  ensure  sterilisation,  and  Gaflky  and  Loffler 
state  that  the  spores  of  some  organisms  are  killed  only 
by  exposure  to  hot  air  at  140°  C.  for  three  hours.  Moreover, 
dry  heat  has  little  power  of  penetration,  and  it  requires 
many  hours  for  the  centre  of  a  mass  of  bedding,  or  the 
like,  to  attain  the  temperature  requisite  for  sterilisation, 
while  some  articles  and  fabrics  are  distinctly  injured  by 
the  prolonged  heating.  The  highest  temperature  which 
can  be  safely  adopted  for  a  dry-heat  disinfector  is  about 
120°  C.,  and  then  if  large  masses  have  to  be  treated  the 
heating  has  to  be  continued  for  from  eight  to  ten  hours. 
A  rise  of  5°  C.  above  this  temperature  is  sufficient  to 
damage  many  woollen  goods,  which  enhances  the  objections 
to  a  dry-heat  disinfector,  as  it  is  difficult  to  keep  the 
temperature  of  a  large  chamber  constant. 


STEAM  DISINFECTION  625 

For  the  reasons  given  above,  disinfection  by  dry  heat 
is  often  impracticable ;  on  the  other  hand,  moist  heat  is 
more  effective,  is  found  to  work  well  in  practice,  and  is 
now  generally  adopted.  In  the  household,  for  articles 
which  cannot  be  burnt,  brisk  boiling  for  an  hour  or  so  will 
suffice. 

Steam  disinfection. — For  public  disinfectors,  steam  under 
pressure — i.e.  at  a  pressure  greater  than  that  of  the  atmo- 
sphere— is  employed.  Steam  under  pressure  has  not  such 
a  deleterious  action  on  articles,  with  the  exception  of 
leather,  as  dry  heat,  while  its  penetrating  powers  are  far 
greater.  By  "  saturated  steam  "  is  meant  steam  at  the 
temperature  at  which  it  can  condense,  and  the  tempera- 
ture of  the  condensation  point  rises  as  the  pressure  increases. 
By  "  superheated  steam  "  is  meant  steam  at  a  temperature 
higher  than  that  at  which  it  can  condense  ;  therefore 
superheated  steam  has  to  be  cooled  down  into  the  state  of 
saturated  steam  before  condensation  ensues.  If  super- 
heated steam  is  used  for  disinfection,  it  loses  heat  by 
conduction,  and  the  rise  in  temperature  of  the  articles 
treated  approximately  corresponds  to  the  fall  in  tem- 
perature of  the  steam.  With  saturated  steam,  on  the 
other  hand,  immediately  it  is  cooled  an  enormous  amount 
of  latent  heat  is  set  free  by  the  change  in  state  from  the 
gaseous  to  the  liquid  condition,  therefore  saturated  steam 
is  a  far  more  efficient  disinfectant  than  superheated  steam. 
These  considerations  should  always  influence  the  choice 
of  a  steam  disinfecting  apparatus  for  efficient  working. 

The  Equifex  disinfector  is  worked  with  saturated  steam 
at  10  Ib.  pressure  (239°  F.).  The  chamber  consists  of  a 
cylinder  of  mild  steel,  made  without  steam  jacket,  so  as 
to  avoid  risk  of  superheating.  The  cylinder  is  lagged  with 
non-conducting  composition  and  wood,  to  reduce  loss  of 
heat  by  radiation,  and,  as  usually  supplied,  is  furnished 
with  separate  doors  for  infected  and  disinfected  articles 

40 


626  A  MANUAL  OF  BACTERIOLOGY 

respectively.  An  arrangement  can  be  supplied  to  prevent 
both  doors  being  opened  simultaneously.  The  Washington- 
Lyons  apparatus,  or  its  modifications,  is  an  elongated 
cylindrical  boiler  with  double  walls,  forming  a  jacket,  and 
a  door  at  each  end.  The  chamber  is  of  sufficient  size  to 
admit  bedding,  and  is  built  into  the  partition  wall  between 
two  rooms,  so  that  each  door  opens  into  a  different  room. 
Into  one  of  the  rooms  the  infected  articles  are  conveyed, 
and  are  placed  in  the  disinfector  as  lightly  packed  as 
possible ;  when  disinfected  they  are  removed  by  the 
opposite  door  into  the  other  room,  thereby  avoiding  all 
chance  of  reinfection.  Steam  at  a  pressure  of  about  20  Ib. 
is  admitted  into  the  jacket  and  then  passes  to  the  inner 
chamber,  the  object  of  the  jacket  being  to  warm  the 
chamber,  and  so  prevent  condensation.  For  the  same 
purpose  hot  air  is  sometimes  injected  beforehand  to  warm 
the  chamber  and  articles,  and  after  the  steam  disinfection, 
can  again  be  injected  for  drying.  The  length  of  time  re- 
quired for  disinfection  does  not  exceed  a  half  to  one  hour. 
In  Thresh's  disinfector  the  steam  is  generated  from  a 
saline  solution  (calcium  chloride),  which  has  a  boiling- 
point0  (105  C.)  higher  than  that  of  water. 

The  thermal  death-point  of  a  number  of  organisms  in  pure 
culture  has  been  determined  by  many  investigators.  Eyre  suggests 
the  following  as  "  standard  conditions  "  for  determining  thermal 
death-points  : 

1.  Length  of  "  time  exposure  "  to  be  ten  minutes. 

2.  Emulsion  to  be  prepared  from  "  optimum  cultivation." 

3.  The  vehicle  in  which  culture  is  suspended  to  be  sterile  salt 
solution  or  sterile  distilled  water. 

4.  Strength  of  emulsion  to  correspond  to  about  1  milligramme  of 
culture  per  cubic  centimetre. 

5.  Bulk  of  emulsion  to  be  not  less  than  3  c.c. 

6.  Emulsion  to  be  contained  in  test-tube  of  1-5  cm.  diameter  with 
walls  1  mm.  thick. 

7.  Emulsion  to  be  exposed  to  moist  heat  in  a  water-bath  regulated 
by  a  delicate  and  accurate  thermo -regulator. 


CHEMICAL  DISINFECTANTS  627 

8.  Broth  cultivations  and  agar  plates  both  to  be  used  in  deter- 
mining the  death  of  the  bacteria,  and  the  period  of  observation  of 
these  cultures  to  be  extended,  when  necessary,  to  seven  or  fourteen 
days.     The  experiments  to  be  repeated  at  least  once. 

9.  Thermal  death-point  to  be  first  roughly  determined  to  within 
5°  C. 

10.  Thermal   death -point   to   be   finally   determined   to   within 
1°  C.,  and  to  be  defined  as  that  temperature  which  causes  the 
death  of  all  micro-organisms  exposed  to  it,  within  the  ten  minutes 
in  these  standard  conditions. 

LIGHT  is  not  used  directly  for  disinfection,  but  indirectly 
in  nature  and  in  our  homes  may  not  be  an  unimportant 
factor.  It  has  previously  been  referred  to  at  p.  23.  Sun- 
light, and  artificial  light  rich  in  violet  and  ultra-violet  radia- 
tions, such  as  that  emitted  by  a  quartz  mercury  vapour 
lamp,  are  efficient  germicides.  The  latter  has  been 
tested  by  Barnard  and  the  writer  with  excellent  results, 
but,  unfortunately,  the  germicidal  rays  have  practically  no 
power  of  penetration  and  are  stopped  even  by  thin  glass. 

DESICCATION,  although  one  of  Nature's  methods  of 
disinfection,  is  not  made  use  of  to  any  extent  by  man 
except  as  an  inhibitory  agent  for  the  preservation  of  many 
articles  of  food.  Shattock  and  Dudgeon  found  that  many 
bacteria,  e.g.  B.  coli.  and  B.  typhosus,  rapidly  succumb  to 
complete  desiccation,  but  B.  pyocyaneus  maintained  its 
vitality  for  two  years  under  these  conditions. 

FILTRATION  is  a  method  of  disinfection  by  exclusion, 
and  in  the  form  of  sand  filtration  and  filtration  through 
porous  porcelain,  as  in  the  Berkefeld  and  Pasteur-Chamber' 
land  filters,  is  made  use  of  for  the  sterilisation  of  water 
and  other  fluids. 

CHEMICAL  DISINFECTANTS. — A  large  number  of  chemical 
substances  variously  known  as  germicides,  antiseptics, 
disinfectants,  deodorants,  etc.,  have  the  power  of  inter- 
fering with,  or  masking  the  results  of,  the  vital  activities 
of  micro-organisms.  Germicides  are  substances  which 


628  A  MANUAL  OF  BACTERIOLOGY 

kill  bacteria  or  germs  ;  antiseptics,  by  inhibiting  bacterial 
development,  prevent  sepsis  or  putrefaction ;  and  by 
"  disinfectant  "  is  meant  a  substance  which  prevents  the 
action  of,  or  destroys,  infective  matters,  while  deodorants 
destroy  or  absorb  foul-smelling  gases  the  result  of  putre- 
factive and  similar  processes.  All  germicides  are  disinfec- 
tant and  antiseptic,  but  many  antiseptics,  though  pre- 
venting or  inhibiting  the  development  of  bacteria,  are  not 
necessarily  germicidal. 

Many  deodorants  act  largely  mechanically,  and  although 
often  not  germicidal,  and  hence  not  ideal  disinfectants, 
are  of  some  value  in  preventing  the  deleterious  and  depres- 
sing effects  of  the  emanations  from  decomposing  organic 
matter.  Such  are  charcoal,  ashes,  dry  mould,  and  peat 
(peat  has  also  a  germicidal  action).  Other  deodorants, 
such  as  quicklime  and  chloride  of  lime,  act  chemically. 

The  germicides  and  antiseptics  may  be  considered  together, 
for  although  many  antiseptics  are  not  germicidal,  all  the 
germicides  in  small  amounts  act  as  antiseptics.  The  prin- 
cipal germicides  and  antiseptics  are  the  halogen  elements, 
the  mineral  acids,  a  large  number  of  metallic  salts,  phenol 
and  many  coal-tar  derivatives,  and  various  organic  bodies 
and  essential  oils. 

Theory  of  chemical  disinfection. — The  theory  of  chemical 
disinfection  is  not  yet  fully  understood.  It  is  probable, 
as  suggested  by  Paul  and  Kronig,  that  the  degree  of  ionisa- 
tion  of  a  solution  may  have  an  important  bearing  on  its 
disinfecting  efficiency. 

Paul  and  Kronig  l  made  a  number  of  experiments  on 
the  M.  pyogenes,  and  spores  of  anthrax,  with  a  view  of 
determining  the  effects  of  various  acids,  bases,  oxidising 
agents,  and  metallic  salts  on  bacteria.  The  salts  of 
mercury,  gold  and  silver  exert  a  marked  germicidal  action, 
strongest  in  the  case  of  mercury,  while  the  platinum  salts 

1  Zeitschr.f.  physikal.  Chem.:  1896,  xxi,  p.  414. 


THEORY  OF  DISINFECTION  629 

are  almost  inactive.  The  efficiency  of  mercuric  chloride 
is  markedly  lessened  by  the  presence  of  sodium  chloride 
or  other  chlorides.  Of  the  oxidising  agents,  nitric,  chromic, 
chloric,  and  permanganic  acids  act  in  the  order  stated ; 
chlorine  has  the  most  powerful  action  of  the  halogens. 
Phenol  acts  better  in  a  5  per  cent,  solution  than  in  higher 
concentrations,  and  the  efficiency  is  increased  by  the 
addition  of  sodium  chloride,  but  diminished  by  the  presence 
of  alcohol,  and  under  the  most  favourable  conditions  it  is 
not  such  a  powerful  germicide  as  mercuric  chloride.  Mer- 
curic chloride  dissolved  in  absolute  alcohol  has  little  or  no 
efficiency,  and  the  addition  of  sodium  chloride  reduces  its 
activity.  Organisms  in  masses  are  less  readily  acted  upon 
by  antiseptics  than  when  they  are  isolated. 

The  efficiency  of  a  germicidal  salt  in  solution  seems  to 
vary  with  its  dissociation.  It  is  believed  that  the  molecules 
of  a  salt  in  solution  are  more  or  less  dissociated  into  con- 
stituent electrified  atoms  or  "  ions,"  and  the  greater  the 
dissociation  the  more  active  will  the  substance  be  as  a 
germicide.  Taking  mercuric  chloride,  bromide  and  cyanide, 
it  is  found  that  the  ionisation  of  the  chloride  is  greater 
than  that  of  the  bromide,  and  this  is  more  ionised  than 
the  cyanide,  and  the  following  results  show  that  the 
germicidal  power  of  the  three  is  in  this  order  :  1 

Number  of 
colonies  which  developed. 

After  After 

Solution.  20  minutes'      85  minutes' 

treatment.        treatment. 

1  mole  HgCl2  in  64  litres     .         7  0 

1     „     HgBr2    „          „      .       34  0 

1     „     Hg(CN)2  in  16  litres          8  33 

Since  the  amount  of  this  dissociation  may  be  greatly 
influenced   by   the   presence   of   other   substances,   much 

1  Findlay,  Physical  Chemistry,  1905. 


630  A  MANUAL  OF  BACTERIOLOGY 

caution  should  be  exercised  in  adding  salts,  etc.,  to  increase 
solubility  or  prevent  precipitation,  as  the  addition  may 
seriously  impair  germicidal  or  antiseptic  power  (see  p.  635). 
The  disinfection  process  is  a  gradual  one.  In  the  early 
stages  of  disinfection  large  numbers  of  organisms  are  killed, 
but  the  rate  of  killing  becomes  slower  and  slower  as  time 
elapses.  Madsen  and  Nyman  and  Miss  Chick  1  have  found 
that  if  the  results  be  plotted,  ordinates  representing  the 
numbers  of  surviving  bacteria,  and  abscissa  the  corre- 
sponding times,  the  points  lie  on  a  logarithmic  curve.  The 
curve  so  obtained,  in  fact,  appears  to  be  similar  in  form 
to  that  of  a  "  unimolecular  reaction,"  and  may  be  expressed 

1  n 

by  the  formula  -     -  log  -  =  K,    where   %   and   n2    are 
t2-t±     &^2 

the  numbers  of  bacteria  surviving  after  times  t±  and  t2 
respectively,  and  K  is  a  constant.  In  the  case  of  disinfec- 
tion of  anthrax  spores  with  phenol,  Miss  Chick  found  the 
mean  value  of  K  to  be  0'44.  In  the  case  of  B.  paratyphosus, 
however,  the  course  of  the  disinfection  is  different  unless 
the  culture  is  very  young,  and  Miss  Chick  concluded  that 
the  older  individuals  are  less  resistant  than  the  younger. 
The  progress  of  heat  disinfection  apparently  follows  the 
same  course.  Miss  Chick  asserts  that  the  act  of  disinfec- 
tion is  a  unimolecular  reaction,  but  it  is  difficult  to  accept 
this  view.  Disinfectants  in  emulsion  tend  to  be  more 
efficient  than  when  in  solution. 

Factors  modifying  disinfectant  action.2 — The  efficiency  of 
a  disinfectant  liquid  partly  depends  on  its  concentration. 
The  rate  of  penetration  into  bacterial  cells  decreases  as 
the  concentration  increases  above  a  certain  limit.  Most 
disinfectants  yield,  therefore,  a  greater  amount  of  disin- 
fectant energy  per  gramme-hour  in  dilute  than  in  strong 

1  Journ.  of  Hygiene,  vol.  viii,  1908,  p.  92  (Summary  and  Bihliog.). 

2  This  section  is  largely  taken  from  Applied  Bacteriology,  Moor  and 
Hewlett,  1907. 


FACTORS  MODIFYING  DISINFECTION      631 

solutions.  In  oil,  glycerin,  or  alcohol,  disinfectants  lose 
some  or  most  of  their  activity.  Spores  in  anhydrous 
glycerin,  oil,  or  vaseline,  are  not  killed  at  a  temperature 
lower  than  170°  C.  acting  for  half  an  hour.1  Of  fats, 
lanolin  alone  seems  compatible  with  disinfectant  efficiency. 
Some  disinfectants  form  an  emulsion  on  the  addition  of 
water,  and  their  efficiency  for  a  given  amount  of  active 
material  may  vary  within  wide  limits  according  to  the 
manner  in  which  they  are  emulsified.  The  temperature 
at  which  the  organism  is  exposed  to  the  disinfectant  has 
a  considerable  influence  on  the  extent  or  rate  of  disinfec- 
tion. Up  to  the  optimum  temperature  at  which  the 
organism  to  be  disinfected  grows  on  the  medium  in  which 
it  is  exposed  the  activity  of  a  disinfectant  may  fall  off 
as  the  temperature  rises,  owing  to  the  increased  vigour 
which  the  organism  derives  from  the  improvement  in 
its  conditions  in  respect  of  temperature.  A  relatively 
small  difference  of  temperature — two  or  three  degrees — 
may  make  an  appreciable  difference  in  the  activity  of  the 
disinfectant,  and  in  the  examination  of  disinfectants  the 
failure  to  remember  this  fact  has  led  to  serious  error. 
Above  the  optimum  a  rise  of  temperature  increases  the 
activity  of  the  disinfectant,  sometimes  to  an  enormous 
extent.  The  same  is  sometimes  the  case  even  at  tem- 
peratures below  the  optimum,  when  the  organism  is  in 
unfavourable  conditions  for  growth.  A  mixture  of  dis- 
infectants in  many  cases  has  a  more  powerful  effect  than 
can  be  produced  by  either  separately  (Chamberland).  The 
resistance  of  bacteria  to  disinfection  by  chemical  agencies 
is  extremely  variable  and  is  also  selective.  Bacteria  of 
one  class  may  be  many  times  more  sensitive  to  one 
disinfectant  than  to  another  when  both  substances 
exert  an  equal  effect  on  bacteria  of  another  class.  The 
presence  of  organic  matter  may  profoundly  modify  the 
1  Bullock  Journ.  of  Hygiene,  xiii,  1913,  p.  168. 


632  A  MANUAL  OF  BACTERIOLOGY 

action  of  chemical  disinfectants,  particularly  those  acting 
by  oxidation,  considerably  reducing  their  efficiency. 

Requirements  for  an  efficient  disinfectant. — The  conditions 
which  should  be  satisfied  by  an  efficient  disinfectant  for 
general  use  are  simple,  but  not  easy  to  obtain.  Because 
a  disinfectant  effect  depends  on  the  strength  of  the  solution, 
the  substance  should  have  an  approximately  definite 
efficiency  for  particular  organisms  in  given  conditions, 
and  for  the  same  reason  it  should  be  permanently  homo- 
geneous. In  practice  disinfectants  must  be  used  with 
water  or  in  an  aqueous  solvent ;  it  should,  therefore,  yield 
a  stable  solution  or  uniform  emulsion  in  all  proportions. 
Because  bacteria  as  presented  for  practical  disinfection 
usually  have  some  organic  coating,  it  should  be  stable  in 
the  presence  of  organic  matter ;  and  as  this  coating  is 
often  of  a  greasy  character,  it  should,  especially  if  intended 
for  use  on  dirty  or  greasy  surfaces,  have  high  solvent  power 
for  grease.  For  use  when  heat  can  also  be  applied,  whereby 
its  activity  is  enhanced,  unless  it  breaks  up,  it  should  be 
stable  at  all  reasonable  temperatures.  These  conditions 
may  be  considered  to  be  indispensable.  It  is  further 
desirable  that  it  should  have  a  sufficiently  high  specific 
efficiency  to  allow  of  its  being  used  in  a  readily  diffusible  dilu- 
tion ;  that  it  should  yield  a  cheap  solution  or  emulsion,  not 
act  on  metals,  and  be  neither  caustic  nor  toxic.  Some  dis- 
fectant  substances  may  now  be  considered  more  in  detail. 

Acids. — All  acids  have  disinfectant  action,  and  their 
relative  values  are  interesting  in  the  respect  that  for  them 
a  general  law  has  been  fairly  well  established  by  Von 
Lingelsheim,  and  confirmed  by  Boer — namely,  that  the  effi- 
ciency varies  with  the  degree  of  acidity.  Solutions  of  acids 
not  of  equal  percentage  concentration,  but  of  equal  acidity, 
have  approximately  the  same  disinfectant  efficiency  what- 
ever may  be  the  acid,  and  whether  it  be  inorganic  or  organic. 

The  acids  have  no  great  practical  application  in  dis- 


ACIDS  AND  ALKALIES  633 

infection.  That  which  has  been  most  commonly  used  is 
sulphurous  acid,  applied  either  direct  from  burning  of 
sulphur  (in  which  case  it  will  also  contain  S03  if  there  is 
sufficient  moisture  to  hold  the  sulphur  dioxide  in  solution) 
or  by  the  use  of  the  liquefied  gas.  It  produces  a  slow 
superficial  disinfection  of  a  weak  and  uncertain  character 
even  under  laboratory  conditions.  Such  experiments 
avoid,  however,  to  a  far  greater  extent  than  is  possible  in 
practice  the  difficulty  of  diffusion,  and  the  unequal  diffusion 
of  sulphurous  acid  in  air  and  its  small  power  of  penetration 
make  it  less  efficient  in  practice.  To  obtain  even  the 
poor  efficiency  which  is  its  maximum  possible  it  is  neces- 
sary for  the  air  to  be  damp  and  the  room  most  carefully 
sealed,  and  in  these  conditions  it  is  often  more  injurious 
to  the  objects  under  treatment  than  to  the  bacteria  against 
which  it  is  directed.  One  of  the  most  efficient  methods  of 
applying  sulphurous  acid  disinfection  is  by  means  of  the 
Clayton  apparatus.  The  gas  is  generated  by  burning 
sulphur  in  a  current  of  air  at  a  high  temperature,  and 
contains,  in  addition  to  S02,  traces  of  higher  oxides  of 
sulphur.  It  is  also  a  very  efficient  vermin-killer,  destroying 
rats,  cockroaches,  bugs,  fleas,  flies,  etc. 

Alkalies  and  soaps. — The  degree  of  alkalinity  of  a  solu- 
tion affects,  but  does  not  by  itself  altogether  determine, 
its  germicidal  power,  which  is  also  dependent  on  the  nature 
of  its  metal.  The  hydrates  of  thallium,  lithium,  barium, 
calcium,  potassium,  sodium,  and  ammonium  have  widely 
different  efficiencies,  roughly  in  the  order  named.  For 
practical  purposes  only  those  of  potassium,  sodium,  and 
calcium  need  be  considered.1  They  exhibit  notably  the 
characteristic  of  all  disinfectants  in  that  they  work  much 
more  vigorously  in  hot  than  in  cold  solution.  It  is  to  the 
hydrates  or  alkaline  carbonates  of  potassium  and  sodium 

1  See  Forrest  and  Hewlett,  Journ.  Roy.  Army  Med.  Corps,  February 
1904. 


634  A  MANUAL  OF  BACTERIOLOGY 

that  the  soaps  owe  such  power  as  they  possess  against 
naked  organisms.  The  relative  efficiency  of  soaps  in 
practical  disinfection  may  be  understated  by  the  results 
of  comparative  experiment  on  laboratory  cultures  because 
the  resistance  of  the  microbe  itself  to  disinfection  by 
chemical  substances,  and,  indeed,  by  other  agencies,  may 
be  small  compared  with  the  resistance  offered  by  the 
envelope  of  grease  or  greasy  dirt,  derived  from  perspiration, 
pus,  fat,  and  the  oily  grime  which  pervades  cities  and  is 
everywhere  caused  by  handling.  A  disinfectant  of  greater 
efficiency  than  soap  on  a  laboratory  culture  may,  therefore, 
be  of  much  less  efficiency  on  an  infection  in  actual  practice. 
Soaps  are  incompatible  with  most  disinfectant  substances, 
but  not  with  all.  Biniodide  of  mercury  can  be  prepared 
with  soap,  and  for  surgical  purposes  is  a  disinfectant  of 
high  value.  The  "  carbolic  soaps  "  of  commerce  are,  for 
the  most  part,  worthless. 

Caustic  lime,  used  generally  as  a  20  per  cent,  milk,  has 
considerable  disinfectant  power,  and  has  been  applied  to 
the  disinfection  of  fseces.  For  this  purpose  care  has  to 
be  taken  to  break  up  any  lumps  of  excreta,  and  whenever 
practicable  a  heat  process,  of  which  the  efficiency  and 
rapidity  may  be  greatly  increased  by  an  alkaline  disinfec- 
tant, is  much  to  be  preferred.  Lime  is  inefficient  against 
»the  more  resistant  organisms,  and  lime-washing  cannot 
be  considered  a  sufficient  precaution  against  them  or 
against  infections,  such  as  those  of  scarlet  fever  and  small- 
pox, of  which  the  exciting  organism  is  unknown. 

Halogens. — The  disinfectant  values  of  dry  chlorine, 
iodine,  and  bromine  are  low.  Both  in  a  dry  and  a  damp 
state  chlorine  is  inconvenient,  and  the  others  are  costly ; 
and  the  use  of  halogens  is  therefore  practically  confined 
to  solutions,  notably  "  chloride  of  lime  "  (a  mixture  of 
calcium  hypochlorite,  hydrate,  and  chloride)  and  hypo- 
chlorite  of  soda  (chloros).  These  have  a  powerful  effect  on 


HALOGENS  635 

laboratory  cultures,  but  in  practice  need  to  be  used  in 
excess  proportionate  to  the  amount  of  organic  matter 
which  may  be  present.  Thus,  for  instance,  a  1  per  cent, 
solution  of  hypochlorite  of  soda  mixed  with  an  equal 
volume  of  urine  loses  the  whole  of  its  available  chlorine 
almost  immediately,  and  becomes  inert  as  a  germicide. 
Where  the  amount  of  organic  matter  is  small,  and  the 
objects  are  not  likely  to  be  injured,  the  hypochlorites  are 
among  the  best  of  known  disinfectants,  provided  they 
are  used  fresh.  The  slow  addition  of  hydrochloric  acid, 
yielding  nascent  chlorine,  increases  the  activity  of  a  hypo- 
chlorite considerably.  A  solution  of  iodine  is  now  used 
for  skin  disinfection  in  surgical  practice.  Iodine  trichloride 
is  a  powerful  disinfectant,  of  which  the  use  has  been 
suggested,  among  other  purposes,  for  the  sterilisation  of 
water.  Nessfield  has  suggested  the  use  of  chlorine  for 
sterilising  water  on  the  large  scale,  and  iodine  for  the 
same  purpose  on  the  small  scale  (p.  599).  Chloride  of 
lime  or  other  hypochlorite  may  be  used  for  sterilising  water 
on  the  large  scale  (p.  600). 

Other  inorganic  substances. — Solutions  of  salts  of  mercury 
exercise  a  powerful  disinfectant  action  in  proportion  to 
the  amount  of  dissolved  metal  which  they  contain.  The 
most  commonly  used  is  the  perchloride  (corrosive  sub- 
limate). Apart  from  its  extremely  poisonous  character, 
it  has  the  disadvantage  of  forming  with  albuminoid  sub- 
stances both  insoluble  and  soluble  compounds  of  little  or 
no  germicidal  value,  sulphuretted  hydrogen  converts  it 
into  the  insoluble  and  inert  sulphide,  and  it  acts  on  some 
metals.  The  addition  of  acids  or  salts  (e.g.  hydrochloric 
or  tartaric  acid  or  sodium  or  ammonium  chloride)  prevents 
or  largely  reduces  the  formation  of  insoluble  compounds  ; 
but  it  does  not  prevent  the  reactions  resulting  in  soluble 
substances,  it  may  reduce  the  germicidal  power,  and  the 
action  of  perchloride  in  the  presence  of  albuminoids  is 


636  A  MANUAL  OF  BACTERIOLOGY 

therefore  very  variable.  The  reduction  in  germicidal 
power  by  addition  of  sodium  chloride  is  well  seen  from  the 
following  results  (Finlay,  loc.  cit.)  : 

Number  of  colonies 

16  litres  of  solution  contained  developing  after  treat- 

ment for  6  minutes. 

1  mole  HgCl2 8 

1     „     HgCl2  +    1  mole  NaCl          .         .  32 

1     „     HgCl2  +    2  moles  NaCl          .         .  124 

1     „     HgCl2  +    4     „     Nad         .         .  382 

1     „     HgCl2  +  10      „     NaCl         .         .  1087 

Extremely  high  values  were  at  one  time  given  for  the 
germicidal  efficiency  of  corrosive  sublimate.  This  is  now 
known  to  have  been  due  to  its  powerful  inhibitory  action, 
traces  of  the  substance  carried  over  into  the  subcultures 
preventing  growth  (see  p.  643). 

The  Local  Government  Board  recommended  the  fol- 
lowing solution  of  corrosive  sublimate  for  disinfecting 
purposes  : 

Corrosive  sublimate  J  oz. 

Hydrochloric  acid         .         .         .  1  oz.  fl. 

Anilin  blue  .          .          .          .          .  5  gr. 

Water 3  gals. 

This  forms  a  solution  of  1-900  nearly ;  it  would  be  pre- 
ferable to  use  1  oz.  of  corrosive  sublimate. 

The  biniodide  is  also  a  powerful  disinfectant  when 
dissolved  in  potassium  iodide.  It  is  not  affected  by 
albuminoids  nearly  as  much  as  is  per  chloride,  and  may 
be  incorporated  with  soap. 

Soluble  silver  salts  are  powerful  disinfectants,  weaker 
than  mercuric  chloride,  but  far  less  sensitive  to  albumi- 
noids ;  in  blood-serum,  for  instance,  silver  nitrate  is 
several  times  as  powerful  as  corrosive  sublimate.  They 
are  incompatible  with  chlorides,  except  in  certain  organic 


FORMALDEHYDE  637 

combinations,  from  which  silver  chloride  is  only  partially 
precipitated.  Silver  salts  are  poisonous,  though  less  so 
than  those  of  mercury. 

Iron  and  zinc  salts  have  been  credited  with  useful 
disinfectant  action  ;  but,  in  fact,  their  value  is  very  small, 
and  no  practical  account  need  be  taken  of  them.  A  very 
strong  antiseptic  power  has  been  attributed  to  copper 
salts,  which,  according  to  some  experiments,  exercise  a 
sufficient  disinfectant  action  on  sporeless  organisms,  such 
as  the  B.  typhosus,  to  enable  drinking  water  to  be  sterilised 
from  such  infections  by  the  small  quantity  of  copper  which 
it  dissolves  (p.  599). 

There  is  some  ground  for  connecting  the  disinfectant 
action  of  metallic  salts  with  a  reducing  action  on  some 
forms  of  protoplasm,  as  pointed  out  by  Loew. 

The  permanganates  have  considerable  germicidal  power 
when  in  strongly  acid  or  alkaline  solution,  but  the  readiness 
with  which  they  are  affected  by  organic  substances  makes 
them  unsuitable  for  practical  use.  Peroxides  and  ozone 
are  open  to  the  same  objection,  and  have  less  disinfectant 
power.  Hydrogen  peroxide  is  used  in  the  Budde  process 
for  sterilising  milk  (p.  615),  and  ozone  has  been  practically 
applied  in  the  sterilisation  of  water-supplies  (p.  600). 

Organic  substances. — The  methane  and  the  aromatic 
series  furnish  the  disinfectants  which  are  most  important 
in  practice. 

Alcohol  itself  possesses  some  disinfectant  power  for 
sporeless  organisms,  but  only  when  absolute  or  in  very 
strong  solution. 

Formaldehyde  is  by  far  the  most  important  of  the 
methane  group.  It  can  be  applied  either  as  a  solution 
(formalin)  or  as  gas.  The  gas  can  be  produced  by  the 
incomplete  combustion  or  oxidation  of  methyl  alcohol, 
by  the  evaporation,  with  or  without  pressure,  or  spraying 
of  formalin,  either  alone  or  mixed  with  calcium  chloride 


638  A  MANUAL  OF  BACTERIOLOGY 

or  glycerine,  by  the  depolymerisation  by  heat  of  the  solid 
polymer  paraformaldehyde,  or  by  mixing  this  substance 
with  potassium  permanganate.  Many  forms  of  apparatus 
have  been  designed  for  the  production  of  formaldehyde  gas 
for  disinfection.  In  any  form  the  gas  seems  to  give  little 
more  than  superficial  disinfection,  and  to  require  precau- 
tions to  ensure  diffusion  throughout  the  atmosphere  of  a 
room.  The  conditions  desirable  for  disinfection  by  for- 
maldehyde gas  are  saturation  of  the  air  with  moisture, 
maintenance  of  a  good  room  temperature,  sealing  of  the 
room,  the  use  of  at  least  60  grm.  of  formaldehyde  per 
1000  cubic  feet  (preferably  more,  up  to  120  grm.),  and  in 
the  case  of  large  rooms  mixture  of  the  gas  with  the  air  of 
the  room,  either  mechanically  or  by  the  provision  of  a 
multiplicity  of  inlets  for  the  gas  into  the  atmosphere.  By 
the  use  of  a  vacuum  formaldehyde  can  be  evaporated  in 
a  closed  chamber  at  temperatures  indifferent  to  many 
substances  which  will  not  stand  steam  at  100°,  and  con- 
siderable penetration  can  be  obtained  (Defries  process). 
As  a  spray  formalin  can  be  used  in  any  ordinary  apparatus. 
Formalin  seems  to  have  a  very  slow  germicidal  action,  for 
tested  by  the  Rideal- Walker  method,  its  carbolic  co- 
efficient is  only  about  0-7  for  the  B.  typhosus.  Yet  2 
per  cent,  formalin  kills  anthrax  spores  in  two  or  three 
days  and  gaseous  formaldehyde  is  similarly  active. 

Of  the  aromatic  series,  the  number  of  substances  and 
preparations  is  extraordinarily  large.  The  standardisation 
of  methods  of  examination  will,  it  is  to  be  hoped,  eliminate 
the  less  efficient. 

The  best  known  is  phenol  (carbolic  acid).  Its  saturated 
solution  contains  about  9  per  cent.  It  is  only  slightly 
affected  by  albuminoids,  and  generally  is  stable  in  the 
presence  of  organic  matter  at  ordinary  temperatures.  Its 
compounds,  when  it  forms  any,  have  themselves  some 
disinfectant  action.  With  acids  this  action  is  usually 


PHENOL  639 

greater  than  that  of  pure  phenol,  with  alkalies  less.  Light 
tends  to  decompose  it,  but  the  efficiency  is  not  affected. 
It  is  poisonous  and  caustic.  For  practical  uses  its  chief 
value  is  as  a  standard,  as  its  disinfectant  value  is  com- 
paratively low,  and  for  spore-bearing  organisms  it  is 
practically  useless.  Like  the  cresols,  its  efficiency  is 
greatly  increased  by  the  addition  up  to  saturation  of 
common  salt  or  hydrochloric  acid.  The  following  results 
well  demonstrate  the  increased  germicidal  power  of  phenol 
by  additions  of  sodium  chloride  (Findlay,  loc.  cit.) : 

Anthrax  spores  treated. 
Number  of  colonies  develop- 

Solution  ing  after  treatment  (days). 

0137 

3  per  cent,  phenol  ....  6300  1390  1260  950 
3  +  1  per  cent.  NaCl  .  5720  1450  1320  360 

3  +  8  per  cent,  NaCl   .       1940       150        50        0 

Probably  the  addition  of  salt  alters  the  distribution  of  the 
phenol  between  the  water  and  the  cells,  the  salt  increasing 
the  concentration  of  the  phenol  in  the  bacterial  cells. 

"  Crude  carbolic  acid  "  consists  mainly  of  cresols  and 
higher  phenols  in  proportions  largely  dependent  on  the 
source  of  the  tar  from  which  they  are  prepared  ;  phenol 
is  nearly  absent  from  it  By  themselves  the  cresols  are 
extremely  insoluble  in  water  ;  in  oil  or  alcohol  they  have 
little  or  no  disinfectant  value.  Cresols  are  much  reduced  in 
efficiency  by  albuminoids.  In  saturated  salt  solution  the  dis- 
infectant value  of  crude  carbolic  acid  is  greatly  increased. 

Ordinarily  neutral  tar  oils  with  no  appreciable  disin- 
fectant value  are  left  in,  or  mixed  with,  tar  distillate,  and 
the  saponified  product  produces  an  emulsion  with  water. 
Innumerable  products  of  this  type  are  made.  Their 
efficiency  varies  not  only  with  their  active  ingredients,  but 
also  with  the  character  of  the  emulsions  which  they  form, 
from  about  the  same  as  that  of  phenol  to  about  three  times 


640  A  MANUAL  OF  BACTERIOLOGY 

as  much.  Commercially  they  are  known  as  soluble  carbolic 
acid,  soluble  creosote,  etc.  Creolin  is  a  type  of  numerous 
preparations  of  the  same  character.  They  are  all  poisonous 
and  sensitive  to  albuminoids.  If  naphthalene  is  present 
in  excess  it  is  deposited  in  cold  weather  on  standing.  Lysol 
is  mainly  a  solution  of  the  cresols  in  fat  or  linseed  oil, 
saponified,  with  addition  of  alcohol.  It  gives  a  clear 
solution  with  water,  having  slightly  less  efficiency  on  naked 
bacteria  than  cresol,  much  superior  solvency  for  grease, 
and  equal  sensitiveness  to  albuminoids.  A  number  of 
proprietary  disinfectants  of  high  germicidal  power  are 
now  to  be  obtained.  Such  are  cyllin,  McDougalPs  M.O.H. 
fluid,  izal,  kerol,  etc.  The  active  agents  appear  to  be 
oxidised  hydrocarbons  without  phenol  and  cresol,  in 
emulsion  in  glue,  soaps,  oils,  etc.,  and  they  are  compara- 
tively non-toxic.  The  active  principle  of  cyllin  is  an 
oxidised  hydrocarbon,  having  a  di-phenyl  nucleus  in  place 
of  the  single  phenyl  present  in  carbolic  acid  ;  it  is  insoluble 
in  water,  hence  for  the  purpose  of  even  distribution  in 
water  it  is  emulsified  with  a  neutral  hydrocarbon  oil. 
The  finished  product  contains  50  per  cent,  of  the  active 
principle,  and  is  free  from  carbolic  acid  and  its  homologues. 
The  active  principle  of  kerol  consists  of  oxidised  hydro- 
carbons with  a  di-phenyl  nucleus  and  contains  no  phenol 
or  cresol.  The  germicidal  efficiency,  expressed  as  the 
carbolic- acid  co- efficient  (p.  645),  of  a  number  of  substances 
is  given  in  the  Table  on  page  641. 

Some  of  the  anilin  dyes,  especially  purified  methyl  violet 
or  pyoctanin,  have  been  claimed  to  be  powerfully  antiseptic 
in  solutions  of  1-500  to  1-1000. 

Chloroform  is  a  powerful  antiseptic,  but  at  least  1  per  cent, 
must  be  present  to  act  as  a  germicide ;  it  is  costly,  and 
not  much  used  as  a  practical  disinfectant,  but  in  bacterio- 
logical and  physiological  chemistry  is  a  useful  antiseptic 
for  preserving  solutions  which  putrefy  easily. 


CARBOLIC  ACID  CO-EFFICIENTS 


641 


lodoform  is  valuable  for  dusting  wounds,  though  its 
penetrating  odour  is  objectionable,  and  has  led  to  the 
introduction  of  many  substitutes.  Its  value  as  an  anti- 

Carbolic  Acid  Co-efficients  obtained  by  the  Rideal- Walker 
Method1  (p.  644) 


Disinfectant. 

Observer. 

Date 
of 
experi- 
ment. 

Organism. 

Carbolic  acid 
co-efficient 
(carbolic  acid 
=  1). 

Absolute  alcohol 

Fowler 

8-05 

B.  typhosus 

0-03 

Boric  acid 

Walker 

10-04 

M 

0  (?) 

Chinosol 

Fowler 

11-03 

M 

0-15 

Chloros  . 

j> 

1-04 

,, 

21-0 

„         (with  50  per 

cent,  urine)  . 

Walker 

7-06 

,, 

8-0 

Copper  sulphate 

ff 

6-04 

M 

0-04 

Cyllin*    . 

Fowler 

11-06 

,, 

14-0 

,,        (with   50   per 

cent,  urine)  . 

n 

5-06 

M 

11-0 

Cyllin      . 

Klein 

5-05 

M.  pyogenes 

9-3 

;> 

Simpson  and 

6-06 

B.  pestis 

34-0 

Hewlett 

Formalin 

Fowler 

3-05 

B.  typhosus 

0-7 

Hydrochloric  acid     . 

Walker 

2-05 

n 

11-0 

Izal* 

Fowler 

3-06 

tf 

11-0 

Kerol*    . 

M 

9-06 

[I 

12-0 

„       (with    50    per 

cent,  urine)  . 

5> 

8-06 

M 

8-5 

Little's  phenyle 

M 

5-04 

2-0 

Lysol 

tj 

2-06 

j} 

2-5 

Mercuric  chloride 

>» 

8-05 

}t 

1000-0 

» 

Walker 

8-05 

400-0 

Potass,  permanganate 

Fowler 

8-05 

?> 

42-0 

(with  3 

per    cent,    organic 

matter) 

Walker 

1-07 

M 

1-0 

Zinc  chloride   . 

» 

1-06 

»> 

0-15 

*  The  germicidal  efficiency  of  these  substances  has  been  increased 
since  the  date  of  the  experiments  recorded,  and  they  now  have  a 
carbolic-acid  co-efficient  of  from  16  to  20-22. 


1  Fowler,  Journ.  Roy.  Army  Med.  Corps,  July  1907. 

41 


642  A  MANUAL  OF  BACTERIOLOGY 

septic  has  been  greatly  discussed  ;  micro-organisms  will 
develop  in  nutrient  media  containing  a  considerable 
proportion,  but  probably  when  in  contact  with  living 
cells  a  decomposition  is  effected,  free  iodine  being  liberated, 
hence  its  value. 

The  essential  oils,  peppermint,  mustard,  doves,  thymol, 
and  menthol,  are  powerfully  antiseptic. 

Disinfectant  powders  at  best  exert  but  a  superficial 
action.  They  act  chiefly  as  deodorants,  but  may  be  useful 
in  preventing  the  breeding  of  flies  in  garbage,  etc. 

It  is  useless  to  add  a  small  quantity  of  disinfectant  to  a  large 
volume  of  fluid  or  solid  ;  the  disinfectant  must  be  added  in  sufficient 
amount  so  that  the  mixture  contains  the  minimum  percentage 
which  has  been  found  by  experiment  to  be  efficient.  For  this 
reason  the  attempt  to  disinfect  sewers,  sewage,  streets,  etc.,  by 
relatively  small  quantities  of  disinfectants  is  useless,  and  the  money 
so  wasted  would  be  far  better  employed  in  providing  more  water 
for  flushing  purposes. 

In  medical  practice,  while  antiseptics  can  be  applied  locally  with 
success  and,  to  some  extent,  for  disinfecting  the  alimentary  tract,1 
no  substance  has  yet  been  discovered  which  can  be  administered 
with  safety  to  such  a  degree  as  to  saturate  the  body,  and  so  exert 
a  general  germicidal  action  in  bacterial  infective  diseases.  Sal- 
varsan,  perhaps,  to  some  extent  possesses  this  power  and  has 
been  used  with  success  in  certain  general  infections,  e.g.  anthrax. 
Protozoa  are  attacked  selectively  by  many  substances,  e.g.  the 
malaria  parasite  by  quinine,  spirochaetes  by  salvarsan,  trypano- 
somes  by  atoxyl,  trypan  red,  etc.,  Piroplasma  canis  by  methylene- 
blue,  etc. 

In  surgical  practice  no  unbiased  observer  can  doubt  the  efficacy 
of  antiseptic  treatment,  but  many  so-called  "  antiseptic  operations  " 
are  marred  by  faults  of  omission  and  commission  which  render 
them  far  from  being  perfectly  antiseptic.  There  has  been  some 
controversy  between  the  advocates  of  "  antise:ptic  "  and  of  "  aseptic" 
surgery.  Undoubtedly  antiseptics  do  diminish  the  vitality  and 
therefore  the  reparative  power  of  the  tissues  and  aseptic  methods 
should  so  far  as  possible  replace  antiseptic  ones.  The  skin  of  the 

1  See  F.  E.  Taylor,  "Intestinal  Disinfection  in  Alimentary  Toxaemia," 
Medical  Prets,  January  14,  1914. 


DETERMINATION  OF  GERMICIDAL  POWER    643 

patient  and  the  hands  of  the  operator  having  been  disinfected  as 
far  as  possible,  no  antiseptic  should  be  permitted  to  come  into 
contact  with  the  wound,  which  may  be  irrigated  with  warm  sterile 
physiological  salt  solution.  A  dry  wound  is  an  important  element  to 
success,  and  a  dry,  sterile,  unirritating  dressing  should  be  employed. 
Instruments,  sponges,  etc.,  may  be  kept  in  sterile  salt  solution  after 
the  preliminary  disinfection — by  heat  (not  sponges)  or  chemicals. 
But  the  aseptic  system  requires  more  care  to  ensure  success  than 
the  antiseptic  one,  and  unless  the  assistants  can  be  Crusted  and 
the  details  rigorously  carried  out,  the  latter  seems  preferable. 


The  Determination  of  the  Germicidal   Power 

For  determining  germicidal  power  on  sporing  organisms  anthrax 
spores  are  generally  used,  on  non-sporing  organisms  cultures  of 
the  B.  typhosus  are  usually  employed. 

(1)  Thread  method. — Sterilised  silk  threads  are  impregnated  with 
sporing  and  non-sporing  organisms,  lightly  dried,  and  then  exposed 
to  the  action  of  the  antiseptic  solution  of  a  known  strength  for  a 
given  time.  After  treatment  the  threads  are  thoroughly  washed 
with  distilled  water  to  remove  the  antiseptic,  and  sown  on  the 
surface  of  agar  or  other  suitable  culture  medium.  If  no  growth 
occurs  the  organisms  are  assumed  to  have  been  destroyed.  As  a 
matter  of  fact,  however,  it  is  extremely  difficult  to  get  rid  of  the 
last  traces  of  the  antiseptic,  which  may  inhibit  growth  although  the 
organisms  may  yet  be  alive,  a  fallacy  which  caused  an  exaggerated 
value  to  be  assigned  to  many  substances — for  example,  corrosive 
sublimate.  The  thread  method  may  still  be  employed,  but  after 
treatment  the  threads  should  be  sown  in  broth,  or,  better  still,  if 
pathogenic  organisms  be  the  subject  of  experiment,  inoculated  into 
a  susceptible  animal.  The  writer  finds  that  in  disinfection  experi- 
ments with  anthrax  spores,  surface  agar  is  a  much  better  medium 
than  broth. 

In  experiments  with  corrosive  sublimate,  by  whatever  method,  the 
last  traces  of  this  substance  must  be  converted  into  the  inert  sulphide 
by  treatment  with  hydrogen  or  ammonium  sulphide. 

(2)  Garnet  method. — Small  garnets  the  size  of  a  pea  are  sterilised, 
soaked  in  a  suspension  or  a  broth  culture  of  the  organism,  removed 
and  dried.  The  garnets  with  the  organisms  attached  are  then 
soaked  in  solutions  of  the  disinfectant  of  known  strengths  for 
various  periods  of  time  ;  they  are  then  removed  from  the  solution 
well  washed  with  sterile  water,  and  finally  placed  in  tubes  of  broth. 


644  A  MANUAL  OF  BACTERIOLOGY 

(3)  Rideal-Walker  or  drop-method. — Moor  first  suggested  that  the 
germicidal  efficiency  of  a  disinfectant  might  be  compared  with  that 
of  a  standard  solution  of  carbolic  acid,  which  has  a  definite  com- 
position, is  stable,  and  can  be  accurately  standardised,  and  Rideal 
and  Walker  devised  an  ingenious  and  simple  method  for  carrying 
this  out.  A  special  test-tube  rack  is  very  convenient  (Fig.  69),  in 
.which  the  lower  tier  has  five  holes  which  hold  three  or  four  tubes 
containing  the  solutions  of  decreasing  strengths  of  the  disinfectant 
tojbe  tested,  and  two  tubes  or  one  tube  containing  standard  carbolic 


FIG.  69. — Test-tube  rack  with  test-tubes  arranged  for  the 
Rideal-Walker  method  of  testing  disinfectants. 

acid  solution  of  known  strength  for  comparison.  The  upper  tier 
has  thirty  holes  in  two  rows  spaced  into  six  sets  of  five  holes 
each.  These  hold  tubes  of  sterile  nutrient  broth  which  are  num- 
bered from  1  to  30.  The  test  is  usually  made  with  a  broth  culture 
of  B.  typhosus,  but  other  organisms  may  be  employed.  The  process 
is  as  follows  :  The  five  tubes  in  the  lower  tier  each  contain  3  c.c. 
of  the  disinfectant  and  carbolic  solutions.  Into  each  in  succession, 
at  intervals  of  half  a  minute,  three  drops  of  the  typhoid  broth 
culture  are  added  with  a  pipette.  Half  a  minute  after  the  last  tube 
has  been  inseminated,  a  loopful  is  taken  from  the  first  tube  and 
inseminated  into  the  first  broth  tube,  and  this  process  is  repeated 
at  half-minute  intervals  until  all  the  broth  tubes  have  been  inocu- 
lated. The  inoculated  broth  tubes  are  then  incubated  at  37°  C. 
for  three  days,  and  the  occurrence  or  not  of  growth  is  taken  as 
indicating  the  killing  or  non-killing  of  the  organism  respectively. 
Obviously  the  first  set  of  five  broth  tubes  inoculated  are  subcultures 


RIDEAL-WALKER  METHOD 


645 


in  which  the  organism  has  been  acted  upon  by  the  disinfectant  and 
carbolic  solutions  for  two  and  a  half  minutes,  the  second  set  for 
five  minutes,  and  so  on.  The  results  (taken  from  an  actual  test) 
may  be  charted  as  follows  : 

B.  typhosus,  24:-hour  broth  culture  at  37°  C. 
Room -temperature  60°  F. 


Disinfectant 

Dilution. 

Time  culture  exposed  to  action 
of  disinfectant  (in  minutes). 

Sub-cultures. 

Period  of 
incubation. 

Tempera- 
ture. 

2J 

5 

7* 

10 

12i 

15 

X 
X 

1-1400 
1-1500 

+ 
+ 

3  days 

37°  C. 

+ 

* 

* 

* 

* 

X 

1-1600 

+ 

+ 

+ 

* 

* 

* 

X 

1-1700 

+ 

+ 

+ 

+ 

* 

* 

Carbolic 

1-100 

+ 

+ 

+ 

* 

* 

* 

=  growth  in  the  sub-cultures. 


=  no  growth  in  the  sub-cultures. 


From  this  it  will  be  seen  that  the  disinfectant  X  in  a  solution  of 
1  in  1600  kills  in  the  same  time  (7|  minutes)  as  carbolic  1  in  100. 
This  result  is  expressed  as  a  coefficient  obtained  by  dividing  the 
strength  of  disinfectant  by  the  strength  of  carbolic  which  kills  each 
in  the  same  time  ;  in  the  present  instance  the  co-efficient  is  YQ00°  = 
16-0,  and  this  figure  is  known  as  the  "  carbolic  acid  coefficient." 

If  nothing  is  known  about  the  strength  of  the  disinfectant,  some 
preliminary  experiments  should  be  performed  with  dilutions  at 
wide  intervals  as  regards  strength  (e.g.  1-100,  1-500,  1-1000, 
1-1500,  1-2000,  etc.),  and  when  the  limit  has  thus  been  approxi- 
mately ascertained,  the  test  is  performed  as  above. 

Precautions  to  be  taken  in  carrying  out  the  test. — (1)  The  culture 
should  be  a  broth  one  about  twenty  to  twenty-four  hours  old,  and 
should  be  free  from  clumps  ;  this  may  be  attained  by  filtration 
through  paper.  Instead  of  adding  drops  of  the  culture  to  the 
solutions,  the  addition  of  0-1  c.c.  of  culture  for  every  cubic  centi- 
metre of  solution  has  recently  been  suggested.  The  writer  regards 
this  amount  as  being  too  large,  and  would  suggest  that  O'l  c.c.  of 
culture  is  sufficient. 

(2)  The  carbolic  acid  (the  crystals  of  which  should  have  a  melting- 
point  of  not  less  than  40-5°  C.)  should  be  kept  in  the  form  of  a 
5  per  cent,  aqueous  solution  standardised  by  the  bromine  method. 


646  A  MANUAL  OF  BACTERIOLOGY 

Failing  this,  the  solutions  may  be  made  with  the  acidum  carbolicum 
liquefactum  of  the  Pharmacopoeia,  which  contains  100  parts  of 
phenol  in  110,  but  is  not  absolutely  constant  in  composition. 

(3)  All  measu  es,  pipettes,  and  test-tubes  used  for  making  dilutions 
should  be  sterile. 

(4)  The  dilutions  of  the  disinfectant  and  carbolic  should  be  made 
with  sterile  distilled  water. 

(5)  The  broth  used  for  culturing  and  sub-culturing  should  have 
the  following  composition : 

Lemco    .          .          .          .          .          .20  grin. 

Peptone  .         .         .         .         .20  grm. 

Salt 10  grm. 

Water 1000  c.c. 

The  medium  should  be  standardised  to  a  reaction  of  +  10  (Eyre's 
scale). 

(6)  The   loop  usecj  for  sulculturing  should    have  an  internal 
diameter  of  3  mm.,  and  be  made  with  platinum  wire  of  27-28 
B.W.G. 

(7)  Growths  in  the  subcultures  should  be  obtained  in  those  taken 
at  not  less  than  two  and  preferably  at  three  of  the  time  intervals 
(2£,  5,  and  7£  minutes)  from  both  the  disinfectant  and  the  carbolic 
solutions  which  correspond. 

(8)  The  temperature  at  which  the  determination  is  made  should 
be  noted,  and  the  strength  of  carbolic  varied  accordingly  (1-100 
for  56°-62°  F.,  1-110  for  62°-67°  F.,  and  1-120  for  67°-73°  F. 
for  B.  typhosus),  or  the  determination  may  be  made  at  a  standard 
temperature  (e.g.  20°  C.)  by  warming  (or  cooling)  the  disinfectant 
and  carbolic  tubes  in  a  water-bath. 

(9)  When  the  organism  does  not  form  a  uniform  culture  in  broth, 
a  suspension  of  an  agar  or  other  culture  must  be  made  in  water 
and  filtered.     Sub-culturing  in  some  cases  (e.g.  with  B.  pestis  and 
B.  anthracis)  must  be  made  on  agar  or  other  suitable  culture  medium. 

The  method  is  an  admirable  one  for  determining  the  relative 
efficiencies  of  disinfectants  on  naked  organisms  in  the  absence  of 
organic  matter.  But  in  practice  disinfection  is  almost  always 
carried  out  in  the  presence  of  organic  matter,  and  various  suggestions 
have  been  made  with  a  view  of  introducing  this  factor  into  the 
test,  for  the  presence  of  organic  matter  may  reduce  the  carbolic- 
acid  coefficient  of  many  disinfectants  (see  pp.  632-642,  and  Table, 
p.  641).  Among  the  substances  suggested  are  urine,  faeces,  2 
per  cent,  suspension  of  dried  and  sterilised  faeces  (Martin  and  Chick), 
and  milk.  Kenwood  and  Hewlett  found  that  the  presence  of  urine 


RIDEAL-WALKER  METHOD  647 

or  faeces  reduced  the  carbolic  acid  coefficient  of  some  proprietary 
disinfectants  to  a  greater  relative  extent  than  that  of  carbolic. 

The  method  is  also  sometimes  somewhat  erratic  in  practice,  and 
a  number  of  determinations  may  be  needed  before  the  strengths  of 
disinfectant  and  carbolic  which  coincide  are  found.  Occasionally 
also  two  strains  of  B.  typhosus  may  differ  widely  as  regards  the 
germicidal  action  of  the  disinfectant  on  them,  while  they  are  prac- 
tically identical  as  regards  the  germicidal  action  of  the  carbolic. 

Woodhead  and  Ponder  have  proposed  a  modification  of  the 
method.  In  this,  B.  coli  is  used  as  the  test-organism  and  bile-salt 
peptone  water  as  the  culture  medium,  a  platinum  spoon  being  used 
for  culturing,  and  more  cultures  at  shorter  intervals  up  to  half 
an  hour  are  made. 

4.  Volatile  disinfectants  may  be  tested  by  moistening  the  wool 
plug  of  an  agar  tube,  inoculating  the  agar,  and  capping  with  a 
rubber  cap,  and  observing  whether  any  growth  occurs. 

5.  Volatile  disinfectants  may  also  be  tested  by  exposing  silk 
threads,  pieces  of  paper  or  fabrics,  splinters  of  wood,  etc.,  impreg- 
nated with  organisms,  some  free,  others  done  up  in  packets  of  cotton- 
wool, in  a  room  or  chamber  of  known  cubic  capacity,  to  the  action 
of  the  gas,  a  known  amount  of  which  is  present  in  the  chamber. 
After  exposure  for  a  given  time,  the  threads  are  sown  in  broth 
tubes,  and  the  tubes  incubated. 

On  the  Rideal- Walker  method,  etc.,  see  Rideal  and  Walker, 
Journ.  Sanitary  Inst.,  vol.  xxiv,  1903,  p.  424  ;  Kenwood  and 
Hewlett,  ibid.  vol.  xxvii,  1906,  p.  1  ;  Firth  and  Macfadyen,  ibid. 
p.  17  ;  Kenwood,  Public  Health,  1908  ;  Fowler,  Journ.  Roy.  Army 
Med.  Corps,  July  1907  ;  Partridge,  Bacteriological  Examination 
of  Disinfectants  ;  Woodhead  and  Ponder,  Lancet,  1909,  vol.  ii. 


FRENCH  WEIGHTS  AND  MEASURES  AND  THEIR 
ENGLISH  EQUIVALENTS 


\fji  (micron) 
1  millimetre 

25  millimetres 
1  centimetre 
2-5  centimetres 
5  centimetres 
1  gramme 
4  grammes 

28  grammes 
1  kilogramme 
0-5  kilogramme 
1  cubic  centimetre 
3£  cubic  centimetres 

28  cubic  centimetres 
568  cubic  centimetres 
1  litre 


0-001  millimetre  (^5Q00  inch,  nearly) 
0-04  (Jg)  inch. 
1  inch. 
0-39  inch. 

1  inch. 

2  inches. 

15£  (15-432)  grains. 

1  drachm  (apothecaries'),  nearly. 

1  ounce  (avoirdupois),  nearly. 

2-2  pounds  (avoirdupois). 

1  pound  (avoirdupois),  nearly. 

16  minims,  nearly  (16'23  minims). 

1  fluid  drachm,  nearly. 

1  fluid  ounce,  nearly. 

1  pint  (f  litre). 

If  pints,  or  35  fluid  ounces,  nearly. 


SOLUBILITIES 

AMOUNT  OF  SUBSTANCE  CONTAINED  IN  10  c.c.  OF  A 
SATURATED  SOLUTION 


Alcoholic  solution  of  methylene-blue     . 
Aqueous  solution  of  methylene-blue 
Alcoholic  solution  of  gentian  violet 
Aqueous  solution  of  gentian  violet 
Alcoholic  solution  of  fuchsin 
Aqueous  solution  of  fuchsin 
Aqueous  solution  of  corrosive  sublimate 


0-068  grm. 
0-646  grm. 
0-442  grm. 
0-175  grm. 
0-292  grm. 
0-066  grm. 
0-507  grm. 


648 


JHD,HODGEND,  O.S 


INDEX 


ABERRATION,  139 
Abiogenesis,  4 
Abscesses,  amoebic,  484 

—  multiple,  226 

—  typhoidal,  355 

Absorption   of  complement,    178, 

183 

Achalme's  bacillus,  427,  565 
Achorion  Schoenleinii,  479 
Acid  Alcohol  in  Gram's  method, 

104 

Acid-fast  organisms,  299 
-  in  milk,  etc.,  340 
Acne,  219,  228,  229 
Actinomyces,  cultivation,  453 

—  varieties,  456 
Actinomycosis,  451 

—  clinical  examination,  456 

—  human,  452 

—  in  cattle,  451 

—  spread  of,  454 

—  staining  of,  452 
Adsorption,  167 
Aerobic  organisms,  21 
Agar,  58.     See  Culture  Media 
Agglutination,  185,190 
Aggressins,  179 

Air,  bacteriology  of,  603 

—  examination  of,  605 

—  of  sewers,  610 

Air- passages,  organisms  of,  570 
Air-pump,  48 
Alcohol,  absolute,  86 

—  formation  of,  35,  385,  471 

—  for  fixing,  85 

—  and  ether  for  fixing,  97 

—  as  an  antiseptic,  365,  637 

—  methylated,  86 


Alessi's  experiments,  365 
Alexins,  174,  181,  200,  204 
Algae,  8,  9 

—  destruction  of,  600 

—  in  water,  602 

Alum  for  purifying  water,  574 

—  method  for  typhoid,  594 
Amboceptor,  174,  175 
Amoeba  buccalis,  482 
Amoeba  coli,  482 
Amoebae,  intestinal,  482 
Amoebic  dysentery,  482 

—  diagnosis  of,  485 
Ammonia  not  pyogenic,  225 

-  production  of,  30,  125 
Anaerobic  cultures,  71 

-  stab,  71 

—  in  nitrogen,  72 

—  Buchner's  tubes,  72 

—  in  vacuo,  72 

—  in  hydrogen,  73 

—  in  formate  broth,  76 

—  in  sulphindigotate  broth,  76 

-  Dean's  method,  76 

-  Frankel's  method,  74 

—  Hamilton's  method,  72 

—  writer's  method,  75 

-  plate,  82 

Anaerobic  organisms,  21,  419 
Analysis  of  yeasts,  466 
Anaphylaxis,  168 
Angina,  Vincent's  269 
Anilin  dyes  as  disinfectants,  640 

—  stains,  99 

—  water,  99 

Animals,  dissection  of,  123 

—  inoculation  of,  122 
Anophelinae,  519 

649 


650 


INDEX 


Anthrax,  251 

-  bacillus  of,  252,  200 

—  diagnosis  of,  262 

—  occurrence  of,  258-261 

—  serum  for,  261 

-  spread  of,  258 

—  symptomatic,  455 

—  vaccine,  262 
Anti- bodies,  149 
Anti-endotoxic  sera,  42,177 
Anti-ferments,  194 
Antigen,  150 

—  test  (syphilis),  501 
Antiseptic      action,        conditions 

modifying,  630 

—  power,  determination  of,  643 

—  treatment,  642 
Antiseptics,  627-643 
Anti-sera,  173 
Anti-serum,  anthrax,  261 

—  cholera,  444 

—  colon,  387 

—  dysentery,  378 

—  gonococcic,  246 

—  hydrophobia,  541 

—  meningococcic,  244 

-  plague,  400 

—  pneumonia,  411 

—  polyvalent,  175 

—  streptococcus,  237 

—  tubercle,  322 

-  typhoid,366 
Antitoxic  constituent,  167 

—  treatment,  160 
Antitoxin,  cholera,  444 

—  diphtheria,  278 

—  tetanus,  424 
Antitoxins,  150 

—  in  normal  blood,  153,  274 
Anti-venin,  164 
Appendicitis,  556 
Archebiosis,  5 

Area  of  dish,  605 
Arthritis,  246,  410,  565,  566 

—  deformans,  566 
Arthus  phenomenon,  171 
Ascitic  fluid  culture  medium,  61 
Ascococcus,  17 
Ascomycetes,  470 


Ascospores  of  peniciUium,  472 

—  of  yeast,  461,  465 

—  of  yeast,  staining,  468 
Aseptic  treatment,  642 
Asiatic  cholera,  433 
Aspergillus  glaucus,  472 

—  fumigatus,  473 

—  niger,  472 
Atrepsy,  206 
Autoclave,  47 
Azotobacter,  33 

BABESIA,  528 

Bacilli,  capsulated,  258 

Bacilli  carriers,  cholera,  438 

-  diphtheria,  273,  286 

—  dysentery,  378 

-  typhoid,  359 
Bacillus,  definition  of,  17 

-  acidi  lactici,  381,  613 

—  acidophilus,  571 

—  acnes,  560 

—  aerogenes  capsidatus,  240,  427 

—  aertryck,  373,  374 

—  albus  variola,  550 

—  alcaligenes,  6,  368,  598 

—  anthracis,  252 

—  anthracis  similis,  256 

—  anthracoides,  256 

—  aquatilis  sulcatus,  598 

—  bifidus,  571 

—  bottle,  479 

-  botulinus,  427 

—  bronchisepticus,  559 

—  buccalis,  460 

—  bulgaricus,  617 

—  butyricus,  35,  432 

—  cadaveris  sporogenes,  431 

—  caniculce,  559 

—  capsulatus,  381 
hominis,  258 

—  cavicida,  388 

—  chauvcei,  429,  431 
Bacillus  cloacce,  389,  584,  610 

-  coli,  379 

communis,  379 

communior,  384 

immobilis,  258 


INDEX 


651 


Bacillus  coryzce,  297 

—  diphtheria,  267 
columbarum,  298 

-  diphtheroid,     273,    287,    298, 

563,  565,  619 

—  dysenteria,  352,  376 

-  enteritidis,  351,  371,  381,  390, 

613,  620 
—  sporogenes,  427,  431 

—  facalis  alkaligenes,  6,  368,  598 

—  fllamentosus,  610,  621 

—  fetidus,  569 

—  fluorescens  liquefaciens,  30,  37, 

203,  239,  363,  581,  610,  621 

—  fluorescens      non  -  liquefaciens, 

581,  621 

—  fluorescens  stercoralis,  363 

—  fusiformis,  296,  19 

—  glanders,  343 

—  grass,  340 

—  icteroides,  373,  546 

—  infantilis,  571 

—  influenza,  415 

—  lactis  aerogenes,  389,  570,  584, 

613 

—  leprce,  333 

—  mallei,  343 

—  megaterium,  621 

—  mesentericus,  30,  610,  621 

—  mist,  340 

—  mucosus  capsulatus,  258 

—  murisepticus,  405 

-  mycoides,  30,  608,  610,  621 
• —  neapolitanus,  388 

-  of  Achalme,  427,  565 
— •  of  black  quarter,  431 

—  of  chicken  cholera,  404 

—  of  Danysz,  373 

—  of  Ducrey,  557 

—  of  Friedlander,  4075  412 

—  of  gastro-enteritis,  371 

—  of  Hofmann,  287 

—  of  hog  cholera,  373 

—  of  Johne,  331 

—  of  Koch  and  Weeks,  557 
• —  of  Laser,  388 

-  of  Lustgarten,  339,  496 

—  of  malignant  oedema,  426 

—  of  Morax  and  Axenfeld,  557 


Bacillus  of  mouse  septicaemia,  405 

—  of  ozsena,  563 

—  of  rabbit  septicaemia,  404 

—  of  rheumatoid  arthritis,  566 

—  of  rhinoscleroma,  566 

—  of  Massol,  617 

—  of  swine  fever,  373 

—  of  swine  plague,  373,  405 

-  of  symptomatic  anthrax,  431 

-  of  syphilis,  496 

-  of  xerosis,  297 

-  Oppler-Boas,  562 

—  paracoli,  374 

-  paradysenterioe,  379 

-  paratyphosus,  351,  371,  374 

—  perfringens,  427 

—  pertussis,  417 

—  pestis,  392 

-  pneumonia,  258,  407,  412 

-  prodigiosus,  36,  250,  621,  622 

-  proteus,  24,  30,  240,  558,  608, 

610,  621 

—  pseudo-anthracis,  256 

—  pseudo-diphtheria,  287 

—  pseudo-dysenteria,  376 

—  pseudo-tuberculosis,  332,  395 

—  psittacosis,  371,  373 

—  putrificus  coli,  30,  420,  571 

—  pyocyaneus,  238,  37,  182,  240, 

541,  558,  559,  561,  627 

—  pyogenes  fetidus,  387 

—  segmentosus,  297 

—  smegmatis,  338,  329 

—  subtilis,  15,  491,  610,  621 

—  suicholera,  373 

-  suipestifer,  351,  373,  374 

—  sulcatus,  598 

—  tetani,  420 

—  timothy  grass,  340 

—  tuberculosis,  301 

—  typhosus,  353 

—  typhimurium,  371,  373 

—  vagina,  571 

—  violaceus,  37,  621 

—  Welchii,  427,    240,    431,    572, 

581,  585,  588,  608,  609,  613, 
617,  618 

—  X.,  546 

—  xerosis,  297 


652 


INDEX 


Bacteria,      action      on      artificial 
sugars,  22 

—  classification  of,  15 

—  conditions  of  life  of,  19 

—  effect  of  electricity  on,  24 

-  effect  of  light  on,  23 

—  effect  of  pressure  on,  23 

-  influence   of   chemical  agents, 

on,  21 

—  influence  of  oxygen  on,  20 

—  influence  of  radium  on,  24 

—  influence    of   temperature   on, 

20 

—  nutrition  of,  19 

—  selective  action  of,  22 

—  structure  of,  9-14 

-  study  of,  66,  118  et  seq. 

-  thermophilic,  20,  608 

—  variation  of,  6,  16 

—  vitality  of  buried,  609 
Bacterial  poisons,  146 

—  products,  38 
Bacteriological      diagnoses.       See 

EXAMINATIONS 

—  microscope,  132 
Bacteriolysis,  174 
Bacteriotropines,  210 
Bacterium,  definition  of,  17 

—  species  of,     See  Bacillus 

—  termo,  30,  621 

—  tumefaciens,  555 
Bacteroids,  32 
Balantidium  coli,  507,  560 
Basidia,  470 
Basidiomycetes,  470 

Bee  disease,  532 
Beer,  466 
Bell- jars,  49 
Beri-Beri,  556 
Berkefeld  filter,  49,  601 
Bird-pox,  206,  553 
Bismarck  brown,  101,  294 
Black  leg,  431 
Black  quarter,  431 
Blackwater  fever,  523 
Blastomycetes,  462 

—  examination,  464 
Blastomycetic  dermatitis,  463 
Bleeding  animals,  125 


Blood  films,  96,  523 
Blood,  centrifuging,126 

—  germicidal  action  of,  199 
—  serum,  60 

-  to  obtain,  60,  125 
Blood-agar,  62,  446 

-  parasites,  staining,  524 
Blue  pus,  238 

Boils,  228 

Borax- methylene  blue,  526 
Bordet- Durham  reaction,  188,  192 
Bordet-Gengou  phenomenon,  183 
Boric  acid,  641 
Bottle  baciUus,  479 
Botulismus,  427 
Bread,  620 

Brilliant-green  agar,  597 
Bromine,  634 
Bronchitis,  557,  248,  413 
Broncho  -  pneumonia,    406,     373, 

413,  416,  417 

Broth,  55.     See  CULTURE  MEDIA 
Brownian  movememt,  11,  130 
Bubonic  plague,  391.     See  Plague 
Buchner's  method,  72 

—  tube,  72 
Budde  process,  615 
Butter,  622 

—  acid-fast  bacilli  in,  340 

CAFFEINE  mixture,  595 
Cahen's  test,  435 
Canary  fever,  549 
Cancer,  554,  232,  462,  486,  511 
Cancrum  oris,  562 
Caps,  india-rubber,  52 
Capsulated  bacilli,  258 
Capsule  of  bacteria,  10 

—  staining,  112 
Carbol-fuchsin,  100 

—  gelatin,  590 

—  methylene  blue,  99 

—  thionin  blue,  100 
Carbolic  acid,  638 

—  crude,  639 

—  coefficient,  645 
Carbuncle,  223 
Carmine  picro-,  101 
Carriers,  bacilli,  359 


INDEX 


653 


Cellulitis,  235 
Centrifuge,  48 

Cerebro- spinal  meningitis,  241 
Chancre,  soft,  557 
Cheese,    diphtheroid    bacillus    in, 
619 

—  tubercle  bacillus  in,  622 

—  spirillum,  449 
Chemotaxis,  202 
Chicken  cholera,  404 
China-green  agar,  597 
Chitral  fever,  549 
Chlamydospores,  471 
Chlamydozoa,  537,  567 
Chloride  of  lime,  634 
Chlorine,  634 
Chloroform,  640 
Chloros,  634 

Cholera  anti-serum,  444 

—  Asiatic,  433 

—  chicken,  404 

—  hog,  372 

—  infantum,  558 

—  red  reaction,  27,  435 

—  spirillum,  433 

—  diagnosis  of,  446 
indole  reaction,  435 

-  in  butter,  622 

-  in  milk,  437,  613,  619 

—  in  oysters,  437 

-  in  soil,  437 

-  in  water,  437,  598 

—  isolation  from  water,  598 

-  pathogenesis,  437 

-  phosphorescence,  440 

—  toxins,  442 

—  vaccine,  444 
Ciliata,  507 
Cirrhosis,  hepatic,  387 
Cladothrix  dichotoma,  460 
Classification  of  bacteria,  15 
Clearing,  108 

Clinical  diagonses.     See  EXAMINA- 
TIONS 
Clostridium  butyricum,  35,  432 

—  Chauvcei,  429,  431 
Clothing,  etc.,  622 

Clove  oil  as  an  antiseptic,  642 

—  as  a  clearing  agent,  108 


Coccidial  disease  in  man,  511 
Coccidium  oviforme,  509 
Cold,  effect  on  bacteria,  20 
Coley's  fluid,  250 
Colitis,  379,  560 
Collodion  sacs,  121 
Colon  bacillus,  379 
—  differentiation    from    typhoid, 
384 

-  isolation  of,  380 

-  isolation  from  water,   590,   et 

seq. 

-  pathogenicity  of,  386 

-  varieties  of,  384,  388 
Comma  bacillus  of  cholera,  433 
Complement,  174,  175 

-  deviation,  178,  183 

-  fixation,  183 
Complementoid,  175 
Condenser,  sub-stage,  139 
Conidia,  469 

Conjugation     in     Hyphomycetes, 

469 

Conjunctive,  organisms  of,  569 
Conjunctivitis,  557 
Conradi-Drigalski  agar,  592 
Contagion,  144 
Copper,  germicidal  action  of,  599 

—  sulphate,  germicidal  action  of, 
599 

Correction  collar,  141 
Corrosive    sublimate    as    disinfec- 
tant, 635,  629 

—  action  on  rubber,  52 
—  for  fixing,  87 

Cover-glass  specimens,  94 

—  of  blood,  97,  523 

—  staining,  106 
Cream,  613 
Creolin,  640 
Cresol,  639 
Crithidia,  487 
Croup,  265 
Cryptococcus,  474 
Culicidse,  518 
Cultures,  anaerobic,  71 

—  hanging-drop,  129 

—  Indian  ink,  81 

—  plate,  76,  82 


654 


INDEX 


Cultures,  preserving,  116 

—  roll,  82 

—  shake,  83 

—  single  ceU,  81,  465 

—  vitality  of,  121 

CULTURE  MEDIA 

Agar-agar,  58 

-  blood,  62,  417,  446,  489 
alkali,  446 

—  brain,  303 

—  brilliant  green,  597 

—  china-green,  597 

—  Conradi-Drigalski,  592 

—  distilled  water,  618 

—  fuchsin,  596 

—  glucose,  59 

—  glycerin,  59 

—  haemoglobin,  63 

—  litmus,  59 

—  malachite  green,  596 

—  maltose,  477 

—  mannite,  33 

—  nasgar,  242 

-  potato  blood,  441 

-  rebipelagar,  592 

—  serum,  62 

—  wood-ashes,  32 
Alkali  albumin,  63 
Ascitic  fluid,  61,  291 
Beer- wort,  57 

Bile  (for  typhoid),  370 
Bile-salt,  590,  591 
Blood-serum,  60 

—  fluid,  61 

—  Loffler's,  61 
Broth,  acid  beef,  54,  64, 

—  ascitic  fluid,  61,  291 

—  egg,  56 

—  formate,  76,  591 

—  glucose,  56 

—  glycerine  beef,  56 

—  Lemco,  56,  646 

—  peptone  beef,  55 

—  sulphindigotate,  76 

—  veal,  56 
Dieudonne's,  446 
Dorset's  egg  medium,  303 
Eggs,  63 

Endo's,  596 


CULTURE  MEDIA  (cont.) — 
Gelatin,  57 

—  beer-wort,  58 

—  carbol,  590 

—  glucose,  58 
Hiss's,  291 
Hydrocele  fluid,  61 
Litmus,  59 
Malachite  green,  596 
Milk,  59 

Neutral  red,  591 
Nitric  and  nitrous,  31 
Pasteur's  fluid,  63 
Peptone  water,  57 

—  Dunham's,  57 
Petruschky's,  385 
Potato,  60 

—  glycerin,  303 
Proskauer-Capaldi,  385 
Standard,  64 
Uschinsky's  fluid,  63 
Whey,  litmus,  385 

Cutaneous  reaction,   tuberculosis, 

330 
-  syphilis,  499 

—  typhoid,  370 
Cultures,  roll,  82 

—  single- cell,  81,  465 
Cystitis,  250,  355,  387 
Cytases,  203 
Cytoryctes  variolce,  551 
Cytotoxins,  185 

DANYSZ  bacillus,  373 

—  effect,  165 

—  rat  vims,  373 
Dark-ground  illumination,  139 
Deneke's  spirillum,  449 
Dengue,  549 

Deodorants,  628 

Dermatitis,  blastomycetic,  463 

—  bullous,  239,  561 
Desiccation  as  a  disinfector,  627 

—  influence  of,  20,  627 
Deviation  of  complement,  178 

—  test,  183 
Dhobie  itch,  479 

Diagnosis,       bacteriological       or 
clinical.     See  EXAMINATIONS 


INDEX 


655 


Diarrhoea    of    infants,    558,    373, 

379,  613 

Dilution  method,  77 
Diphtheria,  265 

-  etiology  of,  266 
Diphtheria,  associated  organisms, 

271 

—  antitoxin,  278 

standardisation  of,  280 

unit  of,  284 

value  of,  285 

—  diagnosis  of,  269,  292 

—  value  of,  270 

-  and  milk,  594 

—  bacillus,  267 

•  acid  formation,  269 

—  fermentation  reactions,  292 

—  in  noma,  562 

—  in  ozsena,  563 

—  in  pyorrhoea,  565 
isolation  of,  266 

-  pathogenic  action,  272 
persistence  of,  270 

-  pseudo,  287 

-  thread  forms,  267,  295 
• toxins,  276 

-  varieties,  267,  272 

—  membrane,  273 

—  in  lower  animals,  275 

—  'of  calves,  298 

—  of  pigeons,  298 
Diphtheritic  roup,  298 

—  neuritis,  273 

-  paralysis,  273,  275,  286 
Diphtheroid  bacilli,  273,  287,  292, 

297,  563,  565,  619 
Diplococcus  crassus,  244 

—  flavus,  244 

—  intracellidaris         meningitidis, 

241 

—  mucosus,  244 

—  pneumonice,  407,  234 

-  rheumaticus,  234,  565,  566 

-  Still's,  244 

Disease,  production  of,  146 
Diseases,  causative  organisms  of, 
556 

—  of  beer,  466 
Disinfectant  powders,  642 


Disinfectants,  627 

Disinfecting     solution     of     Local 

Government  Board,  636 
Disinfection,  623 
Disinfectors,  624-626 
Dissection  of  animals,  123 
Distemper,  559 
Dorset's  egg  medium,  303 
Dourine,  490 
Drepanidium,  531 
Dunham's  solution,  57 
Durham's  tubes,  83 
Dust  in  the  air,  605 
Dysentery,  559 
—  amoebic,  482 

—  bacterial,  376,  239 

—  infusorial,  508 

-  para,  379 

—  pseudo,  376 

—  bacillus,  376,  352 

ECZEMA,  560 
Effluents,  sewage,  610 
Egg  cultures,  63 

—  media,  56,  303 

Ehrlich's  side-chain  theory,  152 

Ehrlich-Biondi  stain,  101 

Eimeria,  509 

El  Tor  vibrios,  441 

Electricity,  effect  of,  on  bacteria, 

24 
Embedding,  gum,  88 

-  paraffin,  90 
Empyema,  355,  410 
Endo's  fuchsin  agar,  596 
Endocarditis,  infective,  228,  231, 

235,  246,  410,  565 
Endotoxins,  39,  40,  147 
Endotoxic  sera,  42,  177 

—  vaccines,  222 
Enrichment  methods,  595 
Entamceba,  482 
Enteritidis  group,  351,  371 
Enteritis,  371 

—  fowl,  404,  447,  511 

—  zymotic,  558 
Enumeration    of    organisms,    80, 

220 
Enzymes,  36 


656 


INDEX 


Eosin,  100 

Epizootic,  lymphangitis,  348,  474 
Eppinger's  streptothrix,  450 
Erysipelas,  236 
Erythrasma,  479 
Esmarch's  roll  cultures,  82 
Ether  and  alcohol  for  fixing,  97 
Euglena,  487 
Evaporation,  49 

EXAMINATIONS,  BACTERIOLOGICAL, 
AND  CLINICAL  DIAGNOSES — 

Actinomycosis,  456 

Agglutination,  190 

Air,  605 

Algae  in  water,  602 

Amoeba  coli,  485 

Anthrax,  262 

Blastomycetes,     pathogenic, 
464 

Butter,  622 

Cheese,  622 

Cholera,  446 

—  in  water,  598 
Ciliated  forms,  508 
Coccidial  disease,  512 
Colon  baciUus,  387 
Complement  fixation,  183 
Diphtheria,  292 

—  in  milk,  618 
Disinfectants,  643 
Dysentery,  485 
Filters,  602 
Flagellated  forms,  508 
Glanders,  349 
Gonorrhoea,  247 
Haemolysis  test,  182 
Hydrophobia,  542 
Hyphomycetes,  474 
Ice  and  ice  creams,  599 
Influenza,  417 
Leprosy,    337 
Malaria,  523 
Malignant  O3dema,  430 
Milk,  618 

Moulds,  474 
Opsonic  index,  214 
Pfeiffer's  reaction,  177 
Phagocytosis,  210 
Plague,  403 


EXAMINATIONS,       BACTERIOLOGI- 
CAL, AND  CLINICAL  DIAG- 
NOSES (cont.) — 
Pneumonia,  414 
Porges'  reaction,  507 
Protozoa,  508 

—  in  water,  602 
Rabies,  542 
Relapsing  fever,  496 
Ringworm,  479 
Sarcina  ventriculi,  249 
Septic  diseases,  239 
Sewage,  610 
Shell-fish,  600 

Smegma  bacillus,  329,  339 
Soil,  609 

Suppuration,  239 
Syphilis,  499 
Tetanus,  425 
Thrush,  474 

Treponema  pallidum,  499 
Trichophytons,  479 
Trypanosomes,  475 
Tuberculosis,  323 

—  (milk),  618 

Typhoid    bacillus    in    water, 
593 

—  fever,  369 
Vincent's  angina,  296 
Wassermann  reaction,  501 
Water,  576 
Watercress,  600 
Welch's  bacillus,  430 
Xerosis,  297 

Yeasts,  468 

—  pathogenic,  464 
Exhaustion  theory,  206 
Eye-pieces,  135,  140 

FARCIN  DES  BCEUFS,  456 
Farcy,  341,  474 
Favus,  479 
Fermentation,  34,  465 

—  acetic  acid,  36 

—  alcoholic,  35 

—  bottom,  466 

—  butyric  acid,  35 

—  lactic  acid,  35 

—  top,  466 


INDEX 


657 


Fermentation  tube,  83 
Ferments,  34,  36 

-  anti-,  194 
Films,  94 

-  blood,  96,  523 

—  interlamellar,  131 
Filters,  49,  601 
Filtration,  49,  574,  601 

—  as  a  disinfector,  627 

-  sand,  574 

';  Finger  and  toe  "  disease,  486 
Finkler-Prior  spirillum,  448 
Fixation  of  complement  test,  183 
Fixing  specimens,  96 

—  tissues,  86 

—  by  alcohol  and  ether,  97 

—  by  corrosive  sublimate,  87 
Flacherie,  532 

Flagella,  11 

Flagella  staining,  114 

Flaginac  reaction,  384,  584 

Flasks,  yeast,  75 

Fleas,  402 

Flies  and  disease,  364,  389 

—  preventing  access  of,  125 

-  tsetse,  490 

Fluid  media,  growths  in,  68 
Food  poisoning,  38,  362,  371,  620 
Foot  and  mouth  disease,  561 
Forceps,  51 
Formalin  for  disinfecting,  637 

—  fixing  tissues,  87 

-  preserving  cultures,  116 

-  preserving  specimens,  116 
Formate  broth,  76,  591 
Fowl  enteritis,  401,  447,  511 
Frambo3sia,  495 
Frankel's  pneumococcus,  407 

—  tube,  74 

Frankland's  method  for  air  ana- 
lysis, 606 

Freezing  microtome,  89 
Friedlander's  capsule  stain,  112 

—  pneumo-bacillus,  412 
Frozen  sections,  88,  108 
Fuchsin  agar,  596 

—  bodies,  554 

-  carbol,  100 
Fumigation,  634,  637 


Fungi,  8,  9,  469,  470 
Fungi  imperfecti,  461,  470 
Fungus  disease,  457 
Fusiform  bacillus,  19,  296,  562 

GAMETES,  481,  515 
Gangrene,  hospital,  223 

-  spreading,  235,  425,  427 
Gartner  group,  351,  371 
Gartner's  bacillus,  371 
Gas  production,  37 

determination  of,  83 

Gastric  juice,  prevention  of  infec- 
tion by,  439,  599 

Gelatin,  57.     See  CULTURE  MEDIA 

—  liquefaction  of,  68 

General  paralysis  of  insane,  274, 

497,  507 

Genital  organs,  organisms  of,  571 
Gentian- violet,  anilin,  99 
Germicidal  action  of  blood,  etc., 

199 

Germicides,  627 
Giant  ceUs,  300,    311,   312,   334, 

346,  451 

Giemsa  stain,  102,  500,  524 
Glanders,  341 
Glanders-like  disease,  349 
Globulin,  cell,  199 
Globulin  of  anti-bodies,  167 
Glossina,  490 
Golding's  bottle,  79 
Gonorrhoea,  244 

—  diagnosis  of,  247 

—  lesions  in,  246 
Gram's  method,  102,  99 

—  Claudius's  modification,  105 

—  Giinther's  modification,  104 

-  thionin,  106 

—  Weigert,  105,  111 
Gram-negative  cocci,  248 
Granules,  metachromatic,  10 
Granuloma,  ulcerating,  495 
Grass  bacillus,  340 

Grease  for  stoppers,  48 
Gregarines,  530 
Griffith's  steriliser,  599 
Grinding  machine,  42 
Grouse  disease,  405,  511 

42 


658 


INDEX 


Guarnieri  bodies,  551,  552 
Gum  for  freezing,  88 


EDEMAMCEBA,  512,  527,  528 
Haematoxylin,  101 

—  iron,  485 
Haemoflagellates,  487 
Haemoglobin  agar,  63 
Hsemogregarines,  530 
Haemolysins,  181 
Haemolysis,  180 

—  test,  182 
Hsemolytic  serum,  181,  184 

—  system,  184 
Haemoproteus,  528 
Haemosporidia,  512 
Halogens,  634 
Halteridium,  528,  515 
Hanging- drop  cultivations,  129 

—  anaerobic,  131 
Haptines,  155 
Haptophore  group,  153 
Heat  as  a  disinfector,  623 
Heat,  dry,  624 

—  moist,  625 
Heidenhain's  iron-haematoxylin, 

485 

Hermann's  tubercle  stain,  327 
Herpes  zoster,  561 
Herpetomonas,  487 
Hesse's  method  for  air  analysis, 

605 

Hiss's  medium,  291 
Hodgkin's  Disease,  332 
Hofmann  bacillus,  287 
Hog-cholera,  372 
Hot-air  steriliser,  45 
Humanus  longus  tubercle  bacillus, 

319 

Hydroa  gestationis,  561 
Hydrocele-fluid   culture   medium, 

61 

Hydrochloric  acid,  635,  641 
Hydrogen  peroxide,  637 
Hydrophobia,  535 
Hypersensitation,  169 
Hyphae,  469 
Hyphomycetes,  469 


Hyphomycetes,  examination,  474 

—  pathogenic,  472 

ICE,  organisms  in,  573,  599 

—  creams,  599 

Identification  of  organisms,  119 
Illumination,  133 

—  dark  ground,  139 
Immersion  lenses,  theory  of,  137 
Immune  body,  174 
Immunity,  195 

—  acquired,  196,  205 

—  atreptic,  206 

—  natural,  196 

—  active,  205 

—  passive,  305 

—  humoral,  202 

—  phagocytic,  202 

—  transmission  of,  206 
Impetigo,  228,  273,  560 
Impression  specimens,  98 
Incubator,  66 

Index,  opsonic,  210,  214 

—  determination  of,  214 
Indian  ink  method,  81 

—  for  syphilis,  499 
Indole,  25 

—  influence  of  culture  medium, 
25 

Infantile  paralysis,  543 
Infection,  144 

—  modes  of,  148 
Infective  process,  147 
Influenza,  415 

—  cold,  248,  297,  417 
Infusoria,  507 

Inoculating  tubes,  method  of,  69 
Inoculation,  intra- venous,  123 

—  of  animals,  122 
Insects  and  disease,  389 
Interlamellar  films,  131 
Intestine,  organisms  of,  570 
Intoxication,  145 
Intracellular  substances,  39,  40, 

140 

Intra- venous  inoculation,  123 
Invertase,  36 
Investigation  of  micro-organisms, 

118 


INDEX 


659 


Iodine,  635 

—  Gram's,  103 

—  trichloride,  635 
lodoform,  641 
Irrigation,  127 

Isolation  of  micro-organisms,  77, 

119 
Izal,  640 

JENNER'S  blood  stain,  102,  524 
Johne's  disease,  331 

KALA-AZAR,  491 

Kerol,  640 

Klebs-Loffler  bacillus,  266 

Koch's  "  comma  "  bacillus,  433 

—  postulates,  147 
Koch- Weeks  bacillus,  557 
Kraus's  test,  435 

LANDRY'S  paralysis,  535,  544 
Lankesteretta,  531 
Laverania  malarice,  521 
Leguminosse,  fixation  of  nitrogen, 

by,  32 

Leishman-  Donovan  body,  491 
Leishman  stain,  102,  524 
Leishmaniosis,  491 
Lenses,  microscopical,  135,  139 

—  immersion,  137 
Leprosy,  333 

—  diagnosis  of,  337 
Leprosy-like  disease  of  rats,  337 
Leptothrix  buccalis,  460 
Leucocytes,  migration  of,  202 

—  in  milk,  616,  619 
Leucocytozoa,  494,  530 
Leucocytozoon  canis,  530 
Leuconostoc,  17 
Levaditi's  stain,  501 
Life-history,  studying,  119,  127 
Life  without  bacteria,  2 
Light  as  a  disinfector,  627 

—  effect  of,  on  bacteria,  23 
Lime  as  a  disinfectant,  634 

—  and  water  purification,  574,  575 
Litmus  media,  59 

Local   Government    Board   disin- 
fecting solution,  636 


Loffler's  methylene  blue,  99 

—  serum,  61 
Loop,  standard,  646 
Luetin,  499 

Lustgarten's  bacillus,  339,  496 
Lymphadenitis,  ovine,  332 
Lymphangitis,  235 

—  epizootic,  348,  474 
Lysins,  150,  174,  181,  185 
Lysol,  640 

MACROGAMETE,  481 
Macrophages,  203 
Madura  disease,  457 
Hadurella,  459 
Mai  de  caderas,  490 
Malachite  green  media,  596 
Malaria,  512 

—  diagnosis  of,  523 
-  parasites,  519-523 

•  mosquito  phase,  516 

species,  519 

Malignant  disease,  554,  232,  462, 
486,  511 

—  oedema,  425 

clinical  examination,  430 

—  pustule,  258 
Mallein,  348 

—  in  diagnosis,  349 
Malta  fever,  567 
Marasmus,  239 
Mastigophora,  487 
Mastoid  disease,  561 
McConkey  stain,  112 

—  media,  590,  591 
McDougall's  fluid,  640 
McLeod's  anaerobic  method,  82 
Measles,  561 

Measurements,  microscopical,  142 
Measures  and  weights,  648 
Meat,  371,  620 

Media,  culture,  54.     See  CULTURE 

MEDIA 

Medical  antiseptics,  642 
Mediterranean  fever,  567 
Meiostagmin  reaction,  193 
Membranous  rhinitis,  273 
Meningitis,  410,  561 

—  cerebro-spinal,  241 


660 


INDEX 


Meningitis,  posterior  basic,  244 
Mercaptan,  25,  37 
Mercuric  chloride,  635 

—  iodide,  636 
Mercury  pyogenic,  225 

-  vapour  lamp,  134,  627 
Merismopedia,  definition  of,  16 
Metachromatic  granules,  10 
Metchnikoff  s  spirillum,  447 
Methylated  spirit,  86 
Methylene  blue,  Loffler's,  99 

—  borax,  526 

—  carbol,  99 

Micrococci,  Gram- negative,  248 
Micrococcus,  definition  of,  16 

—  agilis,  621 

—  bombycis,  532 

—  candicans,  621 

-  catarrhalis,  248,  417 

—  cereus  albus,  231 
flavus,  36,  231 

—  cinereus,  244 

—  deformans,  566 

—  epidermidis  albus,  230,  604 

—  flavescens,  230 

—  gonorrhoea,  244,  566 

—  lanceolatus,  407 

—  Melitensis,561,  188,  248 

—  meningitidis,  241 

—  neoformans,  232,  554 

—  paramelitensis,  569 

-  Pasteuri,  407 

-  pyogenes  aureus,  227 

albus,  229 

citreus,  229 

tenuis,  407 

—  salivarius,  231,  604 

—  scurf,  231,  604 

—  tetragenus,  249 

—  urecR,  30,  36 

—  zymogenes,  231 
Microgamete,  481 
Micrometer,  142 
Micro- millimetre,  143 
Micron,  143,  648 
Microphages,  203 
Microscope,  bacteriological,  132 
Microsporidia.  531 
Microsporon,  Audouini,  475 


Microsporon  furfur,  479 

—  minutissimum,  479 
Microtomes,  89,  92 
Miescher's  corpuscles,  532 
Milk,  612 

-  diphtheria-like       bacilli       in, 

276,  619 

-  examination  of,  618 

-  leucocytes  in,  616,  619 

—  organisms  in,  613 

-  Pasteurisation  of,  614 

-  pathogenic  organisms  in,  613 

—  examination     for     pathogenic 

organisms,  618 

-  sour,  617 

-  standard  for,  617 

—  sterilisation  of,  614 

—  curdling  of,  35,  69,  383,  613 

—  culture  media,  59 

-  and  tuberculosis,  310,  316,  321, 

614 

Moeller's  spore  stain,  114 
Molluscum  bodies,  555 
Morax-Axenfeld  bacillus,  557 
Mosquitoes,  518 

—  and  malaria,  515-519 

—  and  yellow  fever,  547 
Motility  of  organisms,  11,  130 
Moulds,  469 

Mounting,  102 
Mounting  sections,  108 
Mouse  plague,  371 

—  septicaemia,  405 

Mouth,  organisms  of,  460,  570 
Movement,  Brownian,  11,  130 
Much's  tubercle  stain,  327 
Mucor  mucedo,  470 

—  rouxii,  471 

Mucous     membranes,     organisms 

of,  569 
Mumps,  561 
Mussel  poisoning,  38 
Mustard  oil,  642 
Mycelium,  469 
Mycetoma,  457 
Mycetozoa,  486 
Mycoderma,  461 
Mycoses,  472 
Mycosis  tonsillaris,  460 


INDEX 


661 


Mytilotoxin,  38 
Myxomycetes,  486 
Myxosporidia,  531 

NAGANA,  489 

Nasal    mucus    germicidal    action 

of,  570 
Nasgar,  242 
Nastin,  335 
Necrosis,  37 
Needles,  50,  51 
Negri  bodies,  536 
Neisser's  stain,  294 
Neuritis,  diphtheritic,  273 
Nitragin,  33 
Nitrification,  28 
—  stages  in,  30 
—  solutions  for,  31 
Nitrifying  organisms,  isolation  of, 

31 

Nitrobacter,  30 

Nitrogen,  fixation  of,  32 

Nocardia,  459 

Noctiluca,  487 

Noguchi's  method  for  cultivating 

spirochaetes,  497 
Noma,  562 
Nomenclature,  19 
Normal  solutions,  64 
Nosema,  531,  532 
Nose,  organisms,  of,  570 
Nose-piece,  142 
Nucleins,  201 

OBJECTIVES,  135,  139 
Objects,  measurement  of,  142 
(Edema,  malignant,  425 
Oidium  albicans,  474 

—  lactis,  613 
Oil-immersion,  lenses,  137 

Oils,  essential,  as  antiseptics,  642 
Old  age,  571 
Ookinet,  516 

Ophthalmia,  246,  557,  566 
Ophthalmitis,  230 
Ophthalmo-reaction    in    glanders 
349 

—  in  tuberculosis,  330 


Ophthalmo-reaction    in    tvphoid 

370 

Oppler-Boas  bacillus,  562 
Opsonic  index,  210,  219 

-  determination  of,  214 
Opsonins,  210 
Orange-rubin,  101 
Organisms  and  disease,  118    143 

147 
Organisms,  cultivation  of,  66 

—  enumeration  of,  77,  220 

-  identification  of,  119 

-  influence  of  a  mixture  of,  21 

-  isolation  of,  44,  77,  119 

-  of  air,  water,  and  soil,  621 

—  of  air- passages,  570 

—  of  conjunctive,  569 

—  of  genital  tract,  571 

-  of  mouth,  570 

—  of  nose,  570 

—  of  skin,  569 

-  of  stomach  and  intestine,  570 

—  of  urinary  tract,  571 

—  ultra-microscopic,  141 

—  variation  of,  6 

Osmic  acid  fixation,  98,  508 
Osteomyelitis,  227,  228,  235  353 
Otitis,  238,  410,  562 
Oven,  hot-air,  45 
Ozsena,  563 
Ozone,  600,  637 

FAKES'  discs,  583 
Pappataci,  549 
Pappenheim's  solution,  340 
Para-colon  bacillus,  374 
Para- dysentery,  bacillus,  379 
Para-typhoid  fever,  374 
Paraffin,  embedding  in,  90 

—  sections,  92 

Paraffin  sections,  mounting,  109 
Paralysis,   diphtheritic,  273,  275 
286 

—  general,  274,  497,  507 

—  infantile,  543 
Landry's,  535,  544 

Paramecium  coli,  507,  560 
Parasites,  145 
Parotitis,  561 


662 


INDEX 


Parthenogenesis,  481 
Pasteurisation  of  milk,  614 
Pasteur's  fluid,  63 
Peat,   germicidal  action   of,   363, 

437 

Pebrine,  560 
PeUagra,  563 
Pemphigus,  560 
Penicillium  glaucum,  471 
Peppermint  oil,  642 
Peptone  water,  57 
Pericarditis,  246,  410 
Peritonitis,  410,  564 
Permanganates,  599,  637,  641 
Pertussis,  417 
Petri  dishes,  78 
Petri's   method   for   air   analysis, 

606 

Petruschky's  litmus  whey,  385 
Pfeiffer's  reaction,  174,  177 
Phagocytes,  203 
Phagocytosis,  202 

—  estimation  of,  210 
Phenol,  638 
Phlebitis,  226 
Phlebotomus  fever,  549 
Phlogosin,  229 

Phosphorescence,  37,  440,  487 
Phycomycetes,  470 
Physiological  salt  solution,  95 
Picro- carmine,  101 

Pictou  cattle  disease,  387 

Piedra,  479 

Pigment,  formation  of,  36 

Pink  torula,  621 

Pinta,  479 

Pipettes,  51,  53,  214 

Piroplasmata,  528 

Pitfield's  flagella  stain,  115 

Pityriasis,  479 

Plague  antiserum,  400 

—  baciUus  of,  392 

—  diagnosis,  403 

—  epidemiology,  400 
-  pathogenesis,  396 

—  vaccines,  398 
Plasmodiophora  brassicce,  486 
Plasmodium,  512 

—  Kochii,  523 


Plasmodium  malarice,  519 

—  prcecox,  527 

—  vivax,  520 
Plasticine,  52,  82,  215 
Plate  bottles,  79 

—  cultures,  76 

agar,  80 

anaerobic,  82 

gelatin,  78 

silica  jelly,  31 

Platinum  needles,  50 
Plant's  method,  457 
Pleomorphism,  16 
Pleuropneumonia,  141,  407 
Plimmer  bodies,  554 
Pneumobacillus     of     Friedlander, 

412 

Pneumococcus,  Frankel's,  407 
Pneumono-mycosis,  473 
Pneumonia,  406,  371,  373,  391, 413 

—  diagnosis,  414 
-  septic,  235 

Poisons,  bacterial,  37,  146 

—  tolerance  to,  197 
Poliomyelitis,  543 
Porcelain  filters,  49,  601 
Porges'  reaction,  507 
Post-mortems,  123 
Postulates,  Koch's,  147 
Potassium     permanganate,     599, 

637,  641 

Potato,  60.     See  CULTURE  MEDIA 
Powders,  disinfectant,  642 
Precipitins,  194 
Pressure,   effect   of,   on    bacteria, 

23 

Products  of  bacteria,  24 
Proskauer-Capaldi  media,  385 
Proteins,      bacterial,      pyogenic, 

225 

—  germicidal,  199 

—  toxic,  39 
Proteosoma,  527 

Proteus  capsulatus  hominis,  258 

—  mirabilis,  621 

—  vulgaris,  621.     See  B.  proteus 

—  Zenkeri,  621 

—  in  putrefaction,  24,  30 
Protophyta,  8 


INDEX 


663 


Protozoa,  480 

—  action  of  drugs  on,  642 

—  in  water,  602 

Pseudo- diphtheria  bacillus,  287 
Pseudo- diphtheria,  relation  to  B. 

diphtheria,  289 
Pseudo-tuberculosis,  331 
Pseudo  mo  nas,  19,  30 
Psilosis,  564 
Psittacosis,  371,  373 
Psorospermosis,  511 
Pto  mines,  38 
Puerperal  fever,  564 
Pugh's  stain,  294 
Pump,  exhaust,  48 
Purpura,  564 
Pus,  blue,  238 

—  in  milk,  616 
Putrefaction,  24 

Pysemia,  223,  225,  226,  228,  235 
Pyle-phlebitis,  226 
Pyoctanin,  640 
Pyocyanase,  239,  262 
Pyocyaneus  infection,  238,  541 
Pyocyanin,  238 
Pyogenic  organisms,  223,  250 
Pyorrhoea,  565 
Pyrogallic  acid,  73 
Pyrosoma,  528 

QUARTAN  fever,  519 
Quarter  evil,  431 
Quinine  and  malaria,  522 

—  and  tetanus,  422 

RABBIT  septicaemia,  405 
Rabies,  535 

—  diagnosis,  542 

Radium,    effect    of,    on    bacteria, 

24 

Rag-sorter's  disease,  258 
Rat-bite  disease,  565 

—  virus,  373 

Rats  and  plague,  401 
Rauschbrand,  431 
Ray  fungus,  452 

Reaction,     Bordet- Durham,    188, 
192  i 

—  cholera-red,  27,  435 


Reaction,  indole,  25 

—  meiostagmin,  193 

-  Pfeiffer's,  174,  177 

-  Porges',  507 

-  Voges-Proskauer,  389 

-  Wassermann,  501 
Rebipelagar,  592 
Receptors,  154 

—  chemo-,  195,  206 
Relapses,  theory  of,  368 
Relapsing  fever,  494 
Resolving  power,  140 
Retention  theory,  207 
Rheumatism,  565 
Rheumatoid  arthritis,  566 
Rhinitis,  membranous,  273 

—  atrophic,  563 
Rhinoscleroma,  566 
Hhinosporidium  kinealyi,  537 
Rinderpest,  566 
Ringworm,  475 

—  cultivation,  476 

—  examination,  479 
Rocking  microtome,  92 
Roll  cultures,  82 
Romanowski  stain,  102 
Roup,  diphtheritic,  298 
Rubin,  101 

Ruffer  bodies,  554 
Russell's  corpuscles,  554 

SACCHABIMETEB,  84 
Saccharomyces,  461,  465 
Saccharomyces  anomalus,  467 

-  cerevisice,  465,  467 

—  ellipsoideus,  467 
—  litogenes,  462 

—  pastoriamis,  467 

Saliva,  germicidal  action  of,  570 
Salt  solution,  physiological,  95 
Salvarsan,  262,  493,  499,  642 
Sand-fly  fever,  549 
Saprsemia,  225 
Saprophytes,  21 
Sarcina,  definition  of,  17 

—  lutea,  36,  621 

—  ventriculi,  249 
Sarcoma,  462 
Sarcosporidia,  532 


664 


INDEX 


Sarkodina,  481 
Saturation  test,  193 
Scarlet  fever,  533 
Schizomycetes,  8 
Schizophycese,  8 
Sclerotium,  469 
Scour  of  poultry,  540 
Sections,  frozen,  88 

-  paraffin,  90 

-  fixing  to  slide,  93 

—  staining,  108 

—  to  mount,  110 

Sedgwick    and    Tucker's    method 

for  air  analysis,  607 
Sedimentation  test,  192 
Septic  diseases,  223 
Septic  tank  process,  610 
Septicaemia,  149,  223 

-  a,  149 

Sera,  anti-microbic,  173 
— •  antitoxic,  150 

-  polyvalent,  175,  270 
Serum,  culture  medium  of,  60-62 

—  germicidal  action  of,  199 
Serum  disease,  168 
Seven- days'  fever,  549 
Sewage,  609 

Sewers,  air  of,  610 

Shake  culture,  83 

Shell-fish,  examination  of,  600 

—  pathogenic  organisms  in,  362 
Side-chain  theory,  152 

Silica  jelly,  31 
Silkworms,  disease  of,  531 
Silver  salts,  636 

-  pyogenic,  225 
Simulium,  563 
Skatole,  28 
Skatole-carboxylic  acid,  27,  268, 

288 
Skin  diseases,  479,  560 

—  organisms  of,  569 
Sleeping-sickness,  488 
Slides,  cleaning,  95,  114,  524 

—  hollow-ground,  129 
Smallpox,  549 
Smear  preparations,  94 
Smegma  bacillus,  338 

—  staining,  339 


Sodium  bisulphate,  599 
Soil,  608 

Soil,  nitrification  in,  28 
Solubilities,  648 
Solutions,  normal,  64 
Sour  milk,  617 
Species  of  bacteria,  7,  18 
Specimens,     preserving     patholo- 
gical, 116 
Spengler's  tubercle  stain,  326 

—  views  on  tuberculosis,  319 
SpiriUa,  17,  433.    See  also  Vibrio 
Spirillum,  definition  of,  17 

—  choleras  Asiaticce,  433 

-  varieties,  439-442 

—  of     cholera,     isolation     from 

water,  598 

-  of  Tinkler  and  Prior,  448 

—  Metchnikovi,  447 

-  Obermeieri,  494 

-  rubrum,  36,  449 

—  tyrogenum,  449 
Spirochaeta,  18,  493 

-  Duttoni,  494 

—  Obermeieri,  494 

-  pallida,  496 

—  pertenuis,  495 

—  recurrentis,  494 

—  refringens,  496 

—  Vincenti,  296 

—  in  bronchitis,  557 

—  in  cancer,  495 

—  in  dysentery,  559, 

—  in  ulcerating  granuloma,  495 

—  in  ulcers,  449 

—  in  yaws,  495 
Spirochaetosis,  493 
Spironema  pallidum,  496 
Spleen,       germicidal       substance 

from,  200 

—  in  immunity,  204 
Sporangium,  469 
Spore  formation,  14 
Spore  staining,  113,  468 

Spores,  resistance  to  heat,  20,  624 
Sporidium  vaccinale,  552 
Sporotrichosis,  473 
Sporozoa,  508 
Spotted  fever,  241 


INDEX 


665 


Spotted  fever,  of  rocky  mountains, 

546 

Sprue,  564 

Stage,  microscopical,  132 
Staining  methods,  98.    See   under 
respective  names 

—  cover-glass  specimens,  106 

—  capsules,  112 

-  flagelloa,  114 

-  Gram,  102 

—  sections,  110 

-  spores,  113,  468 

Stains,  98.     See    under   respective 

names 

Standard  loop,  646 
Standardisation  of  antitoxin,  280, 
425 

—  of  media,  64 
Staphylococcus,  18 

—  species  of.     See  Micrococcus 
Steam  as  a  disinfector,  625 

-  steriliser,  46 
Stegomyia,  547 
Sterilisation,  45,  624 

—  discontinuous   5 

—  of  cotton- wool,  52 

—  of  glass  vessels,  53 

-  of  milk,  614 
Steriliser,  hot  air,  45 
Steriliser,  Griffith's,  599 

—  writer's,  for  milk,  616 

-  steam,  46,  625 
Still's  diplococcus,  244 
Stimulins,  209 
Stomach,  organisms  of,  570 
Strangles,  237 
Streptococcus,  definition  of,  16 

—  diagnostic  table,  235 

—  anginosus,  234 

—  brevis,  233 

—  conglomerates ,  233,  237,  534 

—  equinus,  235 

—  erysipelatis,  236 

—  foecalis,  234 

—  longus,  233 

—  mzdius,  233 

—  pyogenes,  232 

—  anti-serum,  237 
in  milk,  613,  616,  619 


Streptococcus,    rheunwticus,    234, 
565,  566 

—  vdlivarius,  234,  570,  604 

—  scarlatince,  233,  237,  534 

—  viridans,  236  . 
Streptothrix  infections,  450 
Streptothrix,  acid-fast,  299,  450 

—  actinomyces,  453 

—  Eppingeri,  450,  459 

-  Freeri,  459 

-  leproides,  335 

—  madurw,  458 

-  Nocardii,  452,  459 
Streptotricheae,  450 
Sub-stage  condenser,  139 
Sub-tertian  fever,  521 
Sugars,  resolution  of,  22 
Sulphurous  acid,  633 
Supersensitisation,  169 
Suppuration,  223 

—  clinical  examination,  239 

—  conditions  modifying,  226 

—  due  to  chemical  agents,  225 

—  influence  of  dose,  227 

embolism,  226 

injury,  227 

Surgical  antiseptics,  642 
Surra,  490 

Swine  erysipelas,  405 

-  fever,  372 

-  plague,  373 
Symbiosis,  21 
Symptomatic  anthrax,  431 
Syphilis,  496 

Syringes,  122 

TABES,  505,  507 

Temperature    influence    on    bac- 
teria, 20 
Test-tubes,  50 
Tetanus,  419 

—  animals  susceptible  to,  421 

—  clinical  examination,  425 

-  bacillus,  420 

—  —  associated  organisms,  421 
and  quinine,  422 

-  antitoxin,  424 
—  toxins,  422 
Tertian  fever,  520 

43 


666 


INDEX 


Texas  fever,  529 

Theobald  Smith  phenomenon,  171 

Thermal  death-point  of  organisms 
in  milk,  615 

Thermal  death-point,  determina- 
tion of,  626 

Thermophilic  bacteria,  20,  608 

Thionin,  carbol,  100 

Thrush,  474 

Ticks,  495,  530 

Tinea,  475 

Tinfoil,  52 

Tissue-fibrinogen,  201 

Tissues,  preparation  of,  86 

Tolerance  to  poisons,  195 

Torula,  pink,  621 

Torulae,  461 

Toxins,  39,  153,  162 

Toxoids,  156,  157,  281 

Toxones,  281,  282 

Toxone  effect,  165,  282 

Toxophile  group,  154 

Toxophore  group,  153 

Trachoma,  566 

Treatment,  antiseptic,  642 

-  antitoxic,  160 
Treponema  pallidum,  496 
Trichomonas,  487 

—  species  of,  496 
Trychophytons,  475 
Trypanoplasma,  487 
Trypanosoma,  487 

-  species  of,  488^91 
Tsetse  flies,  490 

-  fly  disease,  489 
Tube,  microscope,  135 
Tubercle  anti-sera,  322 

—  structure  of,  300 

—  bacillus,  301 

agglutination,  324 

Tubercle   bacillus,   avain  variety, 

312,  321 

cultivation  of,  303 

distribution  in  tissues,  311 

mammalian  variety,  312, 

313 

staining  peculiarities,  302 

—  thermal  death-point,  309, 

615 


Tubercle  bacillus,  toxins  of,  308 
in  the  blood,  311 

-  in  butter,  622 

-  in     milk,     316,    321,    614, 

618 
Tuberculin,  Behring's,  307 

—  bacillary  emulsion,  307 

—  cutaneous  reaction,  336 

-  new,  306 

-  old,  304 

—  ophthalmo  reaction,  330 

-  R,  306 

-  reaction  in  actinomycosis,  455 

—  in  leprosy,  334 

—  treatment,  307 

—  veterinary,  331 
Tuberculosis,  299 

—  anatomy  of,  300 

—  avain,  312 

—  bovine,  312,  313 

—  complement  fixation  in,  323 

—  diagnosis  of,  323  et  seq. 

—  disinfection  in,  322 

—  immunity  in,  322 

—  in  the  horse,  313 

—  mammalian,  312 

—  piscian,  312 

—  precipitin  reaction  in,  324 

—  pseudo-,  331 

—  Royal    Commission    on,   314, 

319,  320 

—  Splengler's  views  on,  319 

-  spread  of,  320 
Tuberculous  food,  320 

—  sputum,  staining,  324 

—  tissues,  staining,  327 

-  urine,  329 
Tulase,  307 

Turpentine,  pyogenic.  225 
Twort's  stain,  485 
Typhoid  bacillus,  352 

—  carriers,  359 

—  in  the  blood,  354 

—  in  milk,  613 

—  in  water,  360,  593 

-  serum,  366 

—  survival  of,  359,  360 

—  isolation  from  stools,  370 

—  variation  of,  368 


INDEX 


667 


Typhoid  vaccine,  367 
Typhoid  fever,  352 

—  agglutination     reaction,     356, 

370 

—  and  oysters,  362 

—  and  sewer  gas,  365 

-  diagnosis  of,  369 

—  in  animals,  355 
Typhus  fever,  554 
Tyrotoxicon,  39,  614 

ULCERATING  granuloma,  495 
Ulcerative        endocarditis.        See 

Endocarditis,  infective 
Ulcers,  449 

Ultra- microscopic  organisms,  141 
Ultra-violet  light,  7,  23,  254,  627 
Undulant  fever,  188 
Units,  antitoxin,  284 
Unna's  method,  111 
Urea,  fermentation  of,  30,  36 
Urinary  organs,  organisms  of,  571 
Urine,  colon  bacillus  in,  387 

—  smegma  bacillus  in,  329,  339 

—  tubercle  bacillus  in,  329 

—  typhoid  bacillus  in,  355 
Uschinsky's  fluid,  63 

VACCINES,  dosage  of,  221 

—  endotoxic,  222 

-  prophylactic,  221 

-  sensitised,  222 

—  standardisation,    of,  220 

-  therapeutic,  219 

—  (also      under     individual     or- 

ganisms) 

Anthrax,  262 

Cholera,  444 

Plague.  398 

Typhoid,  367 

Vaccinia,  553 
Vaccinia,  549 
Vaginal  organisms,  571 
Van    Ermengem's    flagella    stain 

114 

Variola,  549 
Vibrio,  definition  of,  17 

-  cholera,  433 

—  Berolinensis.  440 


Vibrio  Danubicus,  440 

-  Deneke,  449 

-  El  Tor,  441 

-  Elwers,  440 

-  Finkler,  448 

-  Ivanhoff,448 

-  Massowah,  440,  441,  443 

-  Metchnikovi,  447 

-  Sanarelli,  463 
Vibrios  of  mouth,  449 
Vincent's  angina,  296, 19 
Virulence,  to  increase,  121 
Visibility,  limit  of,  141 
Vitality  of  cultures,  121 
Voges-Proskauer  reaction,  389 
Volvox,  487 

Vorticella,  507 

WASSERMANN  reaction,  501 

-  in  leprosy,  334,  502 

—  in  malaria,  502 

—  in  scarlatina,  502,  535 

—  in  trypanosomiasis,  502 

-  in  yaws,  495,  502 
Water,  bacteriology  of,  572 

-  number  of  organisms  in,  573 

-  effect  of  sand  filtration,  574 

—  effect  of  sedimentation,  574 

-  effect  of  storage,  573 

—  bacteriological      analysis      of, 

576 

-  pathogenic  organisms  in,  593 

-  colon  bacillus  in,  572,  584,  587, 

590 

—  comma  bacillus  in,  437,  598 

-  sterilisation  of,  599 

—  sterilisers,  599 

-  typhoid  bacillus  in,  360,  593 
Watercress,  examination  of,  600 
Weigert's  law,  155 

-  methods,  105,  111 
Weights  and  measures,  648 
Whooping-cough,  417 

Widal    reaction,    187,     190,    356, 

370 

Wooden  tongue,  451 
Wool-sorter's  disease,  258 
Wright's  capsule,  214 

XEROSIS  bacillus,  297 


668 

YAWS,  495 
Yeasts,  460,  9 

—  analysis  of,  466 
—  isolation,  465 

—  of  fermentation,  465 
-  pathogenic,  462 

Yellow  fever,  546,  373 


INDEX 


ZIEHL  -  NEELSEN    solution, 

100 

Zinc  chloride,  641 
Zoogloea,  10 
Zygospore,  469 
Zygote,  516 
Zymolysis,  34 


JOSEPH  D,  HOOGEN  D, 


BALL ANT YNE    AND    COMPANY 

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